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Human, Eukaryotic And Vitro Associations Of Murine Sec...
INTRODUCTION
In eukaryotes, Usually the UGA acts as a stop codon in transcription but the elongation factor,
known as SELB, has a C–terminal SECIS RNA binding domain and N–terminal Sec–tRNA domain,
inserts the selenocystiene at the stop codon. An SECIS binding protein, known as SBP2 is found in
mammals but it lacks EF functionality. We will describe the in vivo and in vitro associations of
murine SEC–specific EF (eEFSec) element with SBP2 and SECIS in order to correlate their
function in selenoprotein synthesis.
RESULTS
Extensive database search of EST revealed candidates for human, eukaryotic and murine Sec–
specific EF. Murine EST indicates a 583 amino acid protein while the eEF1A shows 462 amino
acids. The protein is homologous to EF and also has portions of eEFsec family. Similar to SELB,
eEFsec lack insertions that are present in eEF1A, responsible to GTP exchange factors interaction.
Homology of c terminal extends both to eukaryote and archeal proteins.
Murine tRNA binding, assessed by nitrocellulose filter binding assay showed high binding with the
two isoforms of Sec–tRNA and lower levels of binding with seryl–tRNA, methionyl–tRNA etc. A
Kd of 0.11 μM was found by scatchard analysis for GTP binding, 3 times less than for GDP binding,
showing that GTP exchange factor is not needed. These results show Sec–tRNA–specific EF.
Co–immunoprecipitation studies were performed to check the hypothesis that binding of SBP2 to
SECIS resulted in recruitment of eEFsec–tRNA. We
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Biotherapeutic Therapy
Biotherapeutic (DNA and Nucleic acid delivery)
Also, biotherapeutic therapy is similar to chemotherapy except that instead of delivering small
molecular drugs (or metabolite ... etc.), biological agents such as DNA, small interfering RNA
(siRNA), proteins, and peptides are delivered to tumor sites to induce apoptosis and cell death. In
cancer, damaged DNA results in defect in protein expression causing deleterious effects. Cancer
may be treated with DNA delivery by replacing the defective genes within cancer cells.
Alternatively, cancer therapy via siRNA works by suppressing the protein expression of damaged
genes. Protein and peptide therapy, on the other hand, operates by attacking specific cell
mechanisms such as disrupting cell adhesion, interfering with angiogenesis, and/or blocking other ...
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In the past, delivery of biotherapeutics has shown limited success due to the immunogenicity caused
by the delivery vectors [154] (were the scientist were using the virus as a vector). Inclusion of
biotherapeutics in NP delivery systems has promise as biocompatible polymer–coated NPs provide
protection against inhibitive immune responses and targeted delivery of these therapeutic agents.
Typically, for gene therapy, NPs are coated with a cationic polymer that encapsulates or binds DNA
or siRNA as the positive charge of this layer allows for favorable electrostatic interactions with
negatively charged nucleic acids [155]. The conjugating of NPs with peptides is other design
considerations for DNA delivery to the cell nucleus for the successful transfection of genetic
material. This is usually accomplished by conjugating peptides that
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Binary Chromatography Lab Report
Afterward, PTEN purified using affinity chromatography. Since PTEN fused with His6 tag, Ni–NTA
affinity chromatography would work to purify PTEN. Ni–NTA is a type of immobilized metal
affinity chromatography that contain coupled Ni2+ nickel chelate forming ligand (imiondiacetic
acid) that immobilized to agarose. The science behind using Ni–NTA affinity chromatography is that
histidine bind specifically and reversibly to the nickel chalet thus washing step will remove other
contaminate and then His–PTEN eluted using imidazole. Bradford assay and SDS–PAGE performed
to quantify and analyze the progress toward PTEN purification. The calculated concentration of the
samples presented in table 8. The Concentration of samples make since having the ... Show more
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A scientist need to demonstrate mammalian cell transfection under very sterile technique since they
grow slowly and affected by contamination resulting in lower expression. In addition, the total
number of mammalian cell should maintain steady and once there is confluent, cells have no more
room to grow, mammalian cell culture should split using digestive enzyme and placed into new
fresh media. To do so, hemocytometer used to count mammalian cell and demonstrate the cell
concentration. Table 11 shows the calculation of mammalian cell concentration and for transfecting
1000,000 cell we need 1.17 ml. Following mammalian transfection, immunoprecipitation processed
for FLAG–PTEN gained from mammalian cells. Anti–FLAG antibody used as ligand just as affinity
chromatography to purify FLAG–PTEN. Latter on, western blotting carried out by transferring the
protein from the completed SDS–PAGE to PVDF membrane as the order in figure 5. After that,
incubation with primary and secondary antibody to visualize PTEN through the chemiliuminescence
reaction as shown in figure 7. The figure present FLAG–PTEN in three samples the FLAG–PTEN
IP, supernatant FLAG–PTEN after IP, and lysate FLAG–PTEN before IP. Lysate FLAG–PTEN
before IP and FLAG–PTEN IP spouse to contain PTEN and that confirm the success of mammalian
transfection. However, the FLAG–PTEN IP showed a smeared band. That may due to high protein
concentration load
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Essay On Proteasome
Recent findings have focused on developing effective proteasome inhibitors as anti–proliferative
agents because previous reports strongly suggest that abnormal or impaired functions of proteasome
aggravate the accumulation of aberrant proteins in cells, which consequently stimulate apoptosis in
cells (Chhangani et al., 2013; Chhangani and Mishra, 2013; Huang and Figueiredo–Pereira, 2010).
Although, the role of proteasome is well–established in clearance of misfolded aggregation of
proteins linked with neurodegeneration and aging,still critical examination of previous studies
suggest that inhibition of proteasome activities can be auseful therapeutic strategy to induce
apoptosis in cancer cells, due to their high metabolic demands and ... Show more content on
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Mitochondrial Membrane Potential Detection JC–1 Kit (BDTMMitoScreen) and FITC Annexin–V–
Apoptosis Detection Kit I (BD PharmingenTM) were obtained from BD Biosciences.
Lipofectamine® 2000, OptiMEM, and JC–1 (5,5', 6,6'– tetrachloro–
1,1',3,3'tetraethylbenzimidazolocarbocyanine iodide) were purchased from Life Technologies and
Molecular Probes respectively. TUNEL assay kit, Dual luciferase reporter gene assay kit and
Proteasome–GloTM assay reagents were obtained from Promega. Anti–ubiquitin, anti–GFP, anti–
20S proteasome, anti–p53, anti–p27, anti–p21, anti–Bax, anti–IkB–α, anti–β–actin and anti–HA
antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase–conjugated
anti–rabbit–IgG and anti–mouse IgG; anti–rabbit, and anti–mouse (IgG fluorescein isothiocyanate
and IgG–rhodamine) were obtained from Vector Laboratories. Plasmid pcDNA™ 3.1 was purchased
from Life Technologies. pcDNA3–EGFP (Addgene 13031), pRK5–HA–Ubiquitin–WT (Addgene
plasmid 17608), pEGFP–Hsp70 (Addgene plasmid 15215) and Luciferase–pcDNA3 (Addgene
plasmid 18964) plasmids were purchased from Addgene.
2.2. Cell Culture, Transfection, Reporter Gene And Cell Viability Assays
COS–7 and A549 cell lines were maintained at 37°C, 5% CO2 in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v)
streptomycin/penicillin (Gibco, Invitrogen). For various transfection and treatment experiments,
cells
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Dendrimers Research Paper
Dendrimers
Dendrimers are 3D polymers with spherical, highly branched structures. Polyesters, polyamines, and
polyamides are commonly used; however, polyamidoamine (PAMAM) dendrimers are the most
frequently used due to their high transfection efficiency. The transfection efficiency is highly
influenced by the size of dendrimers and the type of amine groups that they are composed of. The
primary amine groups participate in DNA binding, so they promote DNA cellular uptake. However,
the tertiary amino groups reinforce the release of DNA into the cytoplasm because they act as a
proto–sponge in endosomes. Transfection efficiency can be enhanced by increasing the size and
diameter of the dendrimers. One example of dendrimer DNA complexes is nitrogen–core
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Endoplasmic Reticulum ( Er ) Stress Response
Abstract
Endoplasmic reticulum (ER) stress response may be mediated by HRV16 nonstructural proteins 2B,
which share similar characteristics to CVB3 2B. To understand whether HRV16 2B induces an ER
stress response, a HRV16 2B fusion protein with GFP were generated and its effects on ATF6a
(activating transcription factor 6a), IRE1 (Inositol–requiring enzyme 1), and PERK (PKR–like ER
protein kinas) expression in BHK cells were determined. The results showed that HRV16 2B
induced significantly high expression of glucose–regulated protein 78, and activated ATF4 and
ATF6 in a time–dependent manner. HRV16 2B also significantly activated the PERK–eIF2α
pathway with marked increased in expression of p–PERK, p–eIF2α, and CHOP. Additionally,
HRV16 2B decreased the expression of p–IRE1 and ATF6 p50. These results demonstrated for the
first time that the PERK–eIF2α signaling pathway, but not IRE1 and ATF6, mediates ER stress in
BHK cells.
Keywords: HRV16, 2B protein, Endoplasmic reticulum stress, PERK Introduction
Human rhinovirus (HRV) infections are major risk factors for asthma and chronic obstructive
pulmonary disease (COPD) [1–4]. The airway epithelial cell is the dominant site of HRV infections
in vivo [5,6], and both upper and lower airway epithelial cells are infected at a similar frequency [7].
Non–enveloped rhinovirus belonging to the Picornaviridae family is spherical in shape, with a
diameter ranging from 28 nm to 30 nm. The viral genome contains a single open reading
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
PEI Transfection Paper
Our project revolved around two major ideas, the first idea that detected the expression of the
Hemagglutinin glycoprotein of Measles virus (MV–H) on the surface of helper virus (Vesicular
Stomatitis Virus) in our tissue culture. The second idea that identified the effect of Axl antibodies on
the penetration processes of VSV–ΔG–MV–H virus The manner of this project represented in three
major stages, the first stage that generated the VSV–ΔG–MV–H pseudovirion. This stage required
to remove the VSV G glycoprotein gene from VSV and replace with gene for the green fluorescent
in helper virus, however, this process was more complex so we skipped this step. The next step that
was extraction the plasmid from the bacteria by following a specific protocol and then started
working with PEI Transfection. ... Show more content on Helpwriting.net ...
For PEI Transfection, we added 1 ml of Trypsin to a T–25 flask of 293T cells and incubate at 37 0C
for 10 minutes. Then, we supplemented 10 ml of DMEM (2.5%), FBS (15), P.S medium to the flack
and finally transferred to dish and put back in CO2 incubator. After 5 days, we diluted plasmid DNA
(ug) in 250 ul of 150mm NaCl in a sterile tube, and diluted 40 ul of PEI into 250 ul of 150 mm NaCl
in second sterile tube. After that, incubated each separate tube for 5 minutes at RT and then mixed
the DNA tube with the PEI tube. We put the tube in incubator for 2–5 minutes at RT, supplemented
dropwise to cells, and put back into incubator. The important steps that was added helper virus
(VSV) to cells and put it in the CO2 incubator; then, collected supernatants that contains our
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Mir34a Essay
MiR34a inhibits Aβ fibril uptake and autophagy flux
Regarding the compromised capability of Aβ clearance in AD, we suspected that miR34a may be an
indispensable player. To validate this, Aβ42 fibrils were prepared from Aβ42 monomer ex vivo and
used to treat glioma A172 and U251 cells. Aβ uptake was blocked by both transient transfection of a
miR34a mimic and stable miR34a overexpression (Supplementary Fig. 1B, Fig. 1A), but enhanced
by anti–miR34a (Fig. 1B), indicating that miR34a suppressed Aβ phagocytosis in these cells. The
fact that enhanced and decreased levels of miR34a correlated with corresponding changes in
intracellular Aβ supports the notion that miR34a may slow down Aβ clearance by affecting its Aβ
uptake and degradation. ... Show more content on Helpwriting.net ...
Since mature autophagosome fuse with lysosomes to become autolysosome in the very late stage of
autophagy, we asked whether miR34a could retard autophagosome–lysosome fusion by examining
the co–localization of EGFP–LC3 and LysoTracker Red, a specific dye for live cell lysosome
labeling [47]. As expected, EGFP–LC3 puncta were dramatically reinforced in miR34a mimic
transfected cells but did not co–localize with LysoTracker Red. This was acting similar to cells
treated with bafilomycin A1 (Baf), which blocked autophagosome–lysosome fusion. In contrast,
E64D treatment induced a remarkable increased EGFP–LC3 puncta, which were well co–localized
with LysoTracker Red (Fig. 1D). Since lysosome–associated membrane glycoprotein 2 (LAMP2) is
also critical for autophagosome–lysosome fusion [48], we examined the effect of miR34a on
LAMP2 expression using WB analysis. MiR34a caused an unexpected increase of LAMP2
(Supplementary Fig. 1C), suggesting that the mechanism by which miR34a blocked
autophagosome–lysosome fusion was not due to LAMP2. As LAMP2 is mostly important for
chaperone mediated autophagy, its increase might be a compensation for the decrease in
macroautophagy. In summary, the above results reinforce that miR34a compromised Aβ degradation
by, at least, blocking autolysosome formation.
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Rhyme Case Study
3.2.1. Creation of the reporter plasmid with VDREs
WT and mutant VDREs fused to a minimal promoter were ordered as premade G blocks with
restriction sites to allow cloning upstream of Firefly luciferase (Method xxxx). 3.2.2. Sequential
digest of SacI and BglII
As there is no buffer in which both SacI and BglII exhibit >50% activity, a sequential digest was
necessary (Method 2.3.). The digest was successful and the resulting cells were then lysed.
3.2.3. Transformation using DH5–alpha cells
Transformation was carried as instructed in Methods2.4.
The cloned VDR was placed in front of the Firefly and Renilla luciferases. A microplate was set up
for the cell transfection and the cells were transfected into HEK293T cells (Method2.5.). A dual
luciferase assay was then run and luciferase ... Show more content on Helpwriting.net ...
This WT stimulation was expected. The difference seen between WT1 and WT2 is due to the use of
two different clones being used. The mutants were found to not be acting as expected. However, the
mutant UGG is higher than the WT UGG. It is possible that is as a result of the mutant having not
been stimulated by calcitriol. Because of this, a fold stimulation plot was produced (Figure 6b). Also
with the UGG mutant codon, it can be seen that the calcitriol is not responsive when transcription
does not stop at the first stop codon.
A final Western Blot was done to analyse the transfected cells.
24 tubes were arranged, each receiving 2.5 μl from each well on the transfection plate to give a total
volume of 10 μl of each example because each transfection has four samples available.
Two separate polyacrylamide gels were run at 100V for 60 mins. The gels were transferred to a
nitrocellulose membrane and treated with primary and secondary antibodies (Methods2.6.). This
was followed with three 5 minute washes with PBS–T, and scanned. The results can be seen in
figure 7 as
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Western Blotting Of Camkii Β And T 287
Western blotting of CaMKIIβ and T–287 phosphorylated CaMKIIβ Western blotting was performed
as described previously (Chapter 3). In short, Proteins were extracted from OLs using RIPA buffer
with protease and phosphatase inhibitors. The concentration of extracted proteins was assessed using
the BCA assay. Target proteins were probed with primary antibodies for CaMKIIβ (1:1000, Life
Technologies) and phosphor–CaMKIIβ (T287) (1:1000, Abcam) at 4°C overnight. Membranes were
incubated with appropriate IRDye secondary antibodies (1:3000, Li–COR, Lincoln, NE) for 1 h at
room temperature, and imaged using an Odyssey Imager (Li–COR). Protein bands were quantified
using Li–COR image studio software. RT–PCR analysis of CaMKII transcription RNAs were
extracted from OLs or cerebral cortex of P2 ICR mice using the miRNAeasy Mini Kit (QIAGEN
Inc. Valencia, CA). Concentration of RNAs from each sample was determined using NanoDrop
ND–1000 spectrophotometer (Thermo Scientific). Equal amount of RNAs were then used as
template to transcribe cDNA using a High capacity cDNA reverse transcription kit (Life
Technologies). Gene–specific primer pairs were designed using NCBI Gene database and primer
design web tool (http://www.ncbi.nlm.nih.gov). The web tool generates a series of primer sets that
fit the parameters defined by users. These parameters include size of the final product and the
primers, whether primers span the junctions between exons, GC contents of the primer etc. For each
isoform
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Lab Report
Results MiR–365b–3p regulates proliferation, migration, invasion and apoptosis of human lung
cancer cells In order to investigate the expression of miR–365b–3p in normal cells and lung cancer
cells, we used real–time PCR to measure the expression of miR–365b–3p in various cells. Results
showed that the expression of miR–365b–3p was significantly lower in lung cancer cells compared
with that in normal cells (Figure 1). After transfection, the expression of miR–365b–3p in COLO
668 cell line was successfully upregulated by miR–365b–3p mimic and inhibited by miR–365b–3p
inhibitor (Figure 2A). Cell viability assay showed that miR–365b–3p suppressed cell viability while
miR–365b–3p inhibitor promoted cell proliferation (Figure 2B). Furtherly, ... Show more content on
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MiR–365b–3p exerts anticancer effect by targeting RAB1B in human lung cancer cells RAB1B
siRNA was used to downregulated the expression of RAB1B. Figure 4A showed that after inducing
with siRNA, the downregulation of RAB1B was confirmed by western blot. According to our cell
viability assay result, transfection with miR–365b–3p inhibited lung cancer cells proliferation,
which was promoted by RAB1B siRNA (Figure 4B). Moreover, siRNA and miR–365b–3p were
both capable of inducing COLO 668 cells apoptosis by activating caspase 3. And according to our
results (Figure 4C, 4D), although there was no statistical significance, the apoptosis–promoting
effect seemed stronger in miR–365b–3p group. We also compared the anti–tumor metastasis effects
between RAB1B siRNA and miR–365b–3p, the results showed both of them significantly reduced
the migration and invasion of COLO 668 cells (Figure 4E, 4F). By comparing with RAB1B siRNA,
we furtherly confirmed the miR–365b–3p exerted anti–tumor effect by targeting RAB1B in human
lung cancer cells. Discussion Various miRNAs are reported to play roles in lung and other cancer.
Based on previous studies, let–7, miR–15, miR–26, miR–29, miR–30, miR–126 and miR–142, have
been reported to have abnormal expression in lung cancer cells compared with normal cells [18]. In
this report, we first time provides evidence that miR–365 exerts anti–cancer
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
How Information Is Processed Through The Brain, Especially...
The authors in this article are trying to better understand how information is processed in the brain,
especially the diseased brain. In order to research this information there first needs to be a precise
method of spatio–temporal recording of electrical activity in larger neuronal circuits and individual
neurons. The authors hypothesized that fluorescent indicators would be useful tools in recording
brain activity because they would allow monitoring of a genetically defined neuronal circuit and
they would not require chemical access to visualize and provide optical recording of brain activity.
Since the current techniques could not meet the authors' requirements to study neuronal activity in
vivo, they sought out to create a voltage ... Show more content on Helpwriting.net ...
Next the authors made a circularly altered Clover GFP by creating termini at the amino acid
positions 145 and 144 and linking its N– and C– termini using the peptide 'GGTGGS' between the
C–terminal (K239) lysine and the original starting methionine. The authors obtained cpsfGFP–
OPT145–144 (abbreviated as cpsfGFP–OPT), which is a circular permutant of a superfolder GFP
variant and which contains 7 mutations that are not present in the original GFP superfolder; it was
made for the use in a split GFP system. The authors then constructed circularly altered superecliptic
pHlurinA227D (cpsepHluorinA227D), creating new termini at the amino acid positions 145 and
linking its original N–and C– termini using the peptide GTGGSAS between the C–terminal lysine
(K239) and a lysine originally near its N terminus (K4). Then authors constructed ASAP1 using the
voltage–sensing domain (VSD) from the Gallus gallus voltage–sensitive phosphatase. The ASAP1
variants were made with the alternate VSDs by inserting cspfGFP–OPT following amino acid G146
(Xenopus laevis), G147 (Danio rerio), or amino acids L203 to G214 (Ciona intestinalis). The
authors isolated the VSD from the VSP by cutting the C–termiminus at the same positions: T183 (G.
gallus), T182 (D. rerio) or S244 (C. intestinalis). Then ASAP1 variants and Archlight Q239 were
cloned between the NheI–HindIII sites of pcDNA3.1/Puro–CAG. The scientists used coexpressed
sensors with a
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Scientific Discovery Of Selective Breeding Essay
Introduction For almost 10 milleniums, with "treatises as much as 2000 years old" giving advice on
selective breeding, humans have carried out genetic manipulation on other organisms for their own
benefit. Humans manipulate the genetics and genetic frequencies of both individual organisms and
entire populations in order to raise the frequency of existing or transgenic traits we find useful or
desirable. Human manipulation occurs for a large number of reasons, including; increasing
profit/productivity in agriculture and farming, creating medical aids for human diseases, increasing
survival chances of the given species and opening pathways for scientific discovery. Two major
methods humans manipulation can be noted; selective breeding, which is the breeding of organisms
within the same species to increase the frequency of existing genes, and transgenesis, the transfer of
genes into the genome of one organism to create new traits, are used. Selective Breeding Selective
breeding is the process in which humans sexually breed animals or plants for traits that are
considered "desirable" for the goal of producing offspring and hopefully; entire lineages, with same
such "desirable trait". Techniques of selective breeding includes marker assisted selection; screening
a genome for specific genes of the parents before actually breeding them, and the original
inbreeding/line breeding which involves breeding organisms with the desired traits with close
relatives to maintain the similar
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Hiv And The Human Immunodeficiency Virus Essay
. Introduction HIV (Human Immunodeficiency Virus) is able to integrate its genome into the host
cell genome during infection (Ebina, Misawa, Kanemura, Koyanagi, 2013). This can lead to AIDS
(acquired immunodeficiency syndrome) if it is left untreated. AIDS is caused by a very efficient and
sneaky retrovirus. HIV can remain dormant for years once its DNA is in the genome of the host
cells. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) are segments of
prokaryotic DNA containing short repetitions of base sequences. Cas9 was the first nuclease
discovered. The focus of the CRISPR/Cas9 is to target a particular disease causing gene and remove
it.
II. CRISPR/Cas9 with HIV cell lines HIV infects CD4 + T cells. The viral particles of HIV keep
their genetic information stored as double–stranded RNA. They use a reverse transcriptase that
converts their genome into double–stranded DNA (Edina, Misawa, Kanemura, Koyanagi, 2013).
During this process the virus can mutate quickly because many errors occur in the HIV genome.
"The DNA is inserted into the host cell genome by integrases which use LTR (long terminal repeat)
sequences on the viral genome to integrate with the host DNA" (Ebina, Misawa, Kanemura,
Koyanagi, 2013). This results in a provirus that can continuously produce new viruses that infect
other immune cells. Doctors can mix different antiretroviral drugs to help with HIV, but once you
stop the treatment the virus
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Accelerated Sensor Of Action Potentials
Accelerated Sensor of Action Potentials 1 The authors were trying to achieve a goal that they
describe as a major goal in the field of neuroscience. They were looking for some method of
reporting electrical activity in neuronal populations, specifically optical reporting. They developed a
voltage sensor that they named Accelerated Sensor of Action Potentials 1 or ASAP1. This allows for
GFP to be inserted into an extracellular loop of a voltage–sensing domain (VSD) and making
fluorescence responsive to membrane potentials. They were looking for ASAP1 to detect a broad
spectrum of membrane potentials, ranging from subthreshold to rapid trains of action potentials. For
this to be the case they had to have the appropriate brightness, dynamic range and kinetics. They
hypothesized that inserting GFP into the S3–S4 loop would allow voltage–induced movements to
interrupt GFP fluorescence. They also hypothesized that a VSD with a shorter loop would increase
the coupling between the voltage–induced movements and GFP barrel distortions. They performed
various tests and concluded that a particular variant of the GFP improved brightness, dynamic range
and expression at the membrane, giving rise to the protein they named ASAP1. From here they
performed tests to show that the VSD that they chose initially was the best choice. There are many
methods that were used in the achieving of this goal. Plasmids are small circular sections of double
stranded DNA that are distinct from a cells
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Methods Of Marker Assisted Selection
Marker assisted selection (MAS) is a method using fluoresces to determine whether there are
mutations in a particular gene. One form of MAS is Fluorescence in situ Hybridization (FISH), in
which the target gene (ADA gene) is denatured (splits into single strands) in a solution containing a
direct DNA probe which has an identical base pair sequence to the ADA gene being analyzed (these
DNA probes are also denatured into single strands). A direct DNA probe is made up of modified
dNTPs which have been altered to contain a fluorophore (a fluorescent chemical compound that has
the ability to re–emit light upon mild excitation), these will bind to the ADA gene strands if there are
no mutations present (i.e. it is a fully functioning gene). If ... Show more content on Helpwriting.net
...
In this time the blastocyst also connects to the endometrial blood vessels in order for it to gain the
nutrients necessary for survival through the blood supply, it is after this that implantation is
considered complete. Medication can also be taken to increase the chances of successful
implantation, such as progesterone, progestins or a GnRH agonist, however these are nonessential
for successful implantation to occur. Implantation is not 100% successful hence the use of these
medications for increased chances perhaps if only a few viable embryos are able to be implanted
Gene Therapy
Gene therapy is a way in which an individual with a preexisting genetic disorder (in this case ADA
deficiency) can be treated. This involves the insertion of a functioning ADA gene into an
individual's cells through the use of viral vectors, to replace the mutated ADA gene that is non–
functioning or functioning incorrectly. In order for this process to be viable multiple techniques are
used, including: restriction enzymes, ligation, PCR and transfection. Through these processes an
individual is able to
As there are 70 known mutations on the ADA gene currently that can cause ADA deficiency, I have
chosen to focus on one specific mutation's gene therapy. This mutation is a substitution mutation
located at the 646th base pair on exon 7, where a G base has been substituted for an A base. In this
case a fully functioning ADA gene is sourced from a donor, in which it will
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Modern Neuroscience, Accurate And Accurate Optical Reporting
In modern neuroscience, accurate optical reporting of electrical activity in defined neuronal
populations can be monumental task. The reason for the enormity of this task is due largely in part
to the lack of a sufficient, effective and accurate optical reporting procedure for activity of the brain.
At this point in time, genetically encoded fluorescent indicators seem to be the answer for accurate
and effective optical reporting, largely because they allow monitoring of defined neuronal circuits
and do not require any chemical access. Ideally, these indicators would produce large fluorescent
responses, allowing imaging on how input signals are processed into output signals by neurons. In
addition, these ideal indicators would have ... Show more content on Helpwriting.net ...
ASAP1 was developed by inserting a circularly permuted green fluorescent protein into a voltage
sensing extracellular domain. It's believed that this design will have sufficient brightness, faster
kinetics, as well as a dynamic range of detection of neuronal activity needed to make this ASAP1
design the ideal fluorescent based reporter for optical reporting of brain activity. This hypothesis
was tested extensively in a laboratory setting to prove the claim. Numerous standard laboratory
techniques and instrumentation was used to obtain the forthcoming results. The ASAP1 expression
plasmids were created by standard molecular biology methods, and then verified through cloned
fragment sequencing. A plasmid was used for multiple reasons, the most prominent being that
plasmids are very easy to manipulate to express a desired genetic element. In addition, they are very
stable as well as self–replicating once initially constructed. HEK293A cells were used in
experimentation and transfected with the ASAP1 plasmid. The HEK293A cells, which are very
popular for transfection, were chosen as they are easy to reproduce, maintain, and have a high
efficiency of transfection. HEK293A cells also have minimal processes that need to be
accomplished in order to preform voltage–clamp experimentation. Transfection is simply the
process by which
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Human, Eukaryotic And Vitro Associations Of Murine Sec...
Gene Therapy Safety
Efficacy and Safety of Gene Therapy
Zack Martin
What is Gene Therapy? A new form of medicine with the potential to save and improve millions of
live is gene therapy, or gene replacement therapy. Gene therapy is the use of DNA as a
pharmaceutical agent used to treat or cure diseases or disorders caused by defective or missing
genes. There are three methods of gene therapy: replacing a mutated gene with a healthy copy of the
same gene, inactivating a gene that is not functioning properly, or introducing a completely new
gene to help the patient fight a disease. Gene therapy is a promising treatment option, however, the
technique has yet to be perfected and is still dangerous and experimental.
Replacing a Mutated Gene with a ... Show more content on Helpwriting.net ...
The first method is to attach the gene to a virus. This is done by removing the functional group of
the virus that harms the body and attaching the gene that is to be delivered into the cell. Retroviruses
and adenoviruses are the two types of viruses used in gene therapy. Retroviral vectors are used
because it inserts its own DNA into the host's DNA. There are two ways to use retroviral vectors in
gene therapy, introduce the vector intravenously, or removing the patient's tissue, inserting the
vector in a lab setting, then replacing the tissue. One of the flaws using the retroviral vector method
in gene therapy is that integrase enzyme(responsible for integrating the retroviral DNA into the host
cell) can insert the gene in any position in the genome. If the gene is place in the middle of another
functional sequence, it would cause a malfunction of that particular gene. Not all gene sequences
have a significant function, but there is still considerable risk in the retroviral vector method.
Although it can be dangerous, this is most commonly used in patients with a small chance of
survival, so the risk is not as
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Tuskegee Syphilis Study
1. What public health measures were widely used prior to the Sanitary Awakening? Before the
sanitary awakening, the health measures used were mainly isolation techniques. Individuals were
left in sectioned off areas to die or let their illness progress until the 1900s. In the 1900s during the
Sanitary Awakening health officials start looking at disease by environmental conditions. 2. In what
year did human population growths peak? In 1960 worldwide population growth reached an all time
peak of 2.0. At this point demographers brought up the issue of carrying capacity.
3. The mean number of children born per woman in a particular population ranges between 1 and
10. This indicator is called what? Total fertility refers to the mean number ... Show more content on
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Local health departments are typically involved in providing preventative health services,
communicable disease control programs, and community health education, among other activities.
Which of the core functions of public health does this represent? The core function being
represented is assurance. At this core function people are linked to personal health services needed,
the medical workforce is checked to be competent, and the community is educated and informed of
health issue that may arise. In addition, education and community health problems are identified and
solved.
16. The Tuskegee Syphilis Study took place during which period of U.S. history? The Tuskegee
Syphilis Study took place from 1932 to 1971 thus referring to the ears of the great depression and
World War II.
17. What is a practice that best illustrates the principal of "Ethical Relativism?" Ethical relativism is
the idea that deciding if an action is right or wrong depends on ones own societal normality. The
practice of medicine demonstrates this principal. A doctor in one country may see giving a
handshake as a welcome as right while in other countries this may not be considered the same.
18. World reaction to atrocities committed under Nazi Germany Led to what document? World
reaction led to the development of the consent
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Rat Brain Case Study
1000 µg rat brain lysate was incubated with 50 µg construct overnight on a shaking incubator at
4°C. Samples were loaded on a polyacrylamide gel and processed for immunoblotting (see
appendix)
Immunoblotting for sacsin and JIP3 interaction
Primary Sacsin and JIP3 antibodies (Santa Cruz cat. sc–515118 and sc–46663) raised in mice were
added in a 1:3000 and 1:2000 ratio respectively to the corresponding membrane sections and
incubated on a shaking incubator for 1 hour. Next, excess antibody was removed by washing 3 times
with TBST. Secondary goat anti mouse HRP antibody (Cedarlane Laboratories cat. 115–035–146)
was added in a 1:4000 ratio to 20 ml of 5% BSA in TBST. The membranes were washed 3 times
with TBST. To image the proteins, 3 ml of ... Show more content on Helpwriting.net ...
After three hours, the coverslips were carefully washed with 1X PBS at 37°C, and fixed for 20
minutes with 4% Paraformaldehyde in PBS.
Immunofluorescence on HeLa cell lines
The coverslips were washed with 1X PBS. Permeabilized for 5 minutes with 500 µl PBS + 0.2%
Triton X–100, then washed again with 1X PBS. The cells were blocked for 1 hour with 2% BSA in
PBS. Then the cells were incubated for 1 hour with primary LAMP1 antibody raised in mice at a
concentration 1:50 (cat. ab25630 Abcam), diluted with 2% BSA in PBS. The coverslips were
washed 3 times with 1X PBS and incubated for 45 minutes with secondary Alexa Flour 647 goat
anti mouse antibody (cat. A21236 Invitrogen), at a concentration of 1:500, diluted in PBS + 2%
BSA. The coverslips were then washed 3 times with 1X PBS. The coverslips were mounted using
DAKO mounting media (cat. S3023), and imaging of the cells was performed using an LSM 710
(ZEISS) confocal fluorescence microscope.
Results, add table for results but remove from text, no methods here at all, controls
JIP3 interaction with sacsin domains
To find the critical binding region of sacsin which interacts with JIP3, six well–characterized N–
terminally GST–tagged sacsin deletion constructs, which include the three SIRPTs, DNAJ–HEPN,
UBE3A binding site, and UBL domains (Fig.1), were expressed in a bacterial system and used in
pulldown assays to test for JIP3 binding. These domains
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Dna Damage, Repair, And Dna Methylation Essay
Introduction:
Modification of damaged DNA seems to be an understudied subject, there is much to understand on
the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by
methylation but much of it is affected on a gene when silenced. When epigenetic modification has
been implicated with cancer and aging it causes DNA methylation to also have an impact on the
double strand of DNA analysis. Modification as such provoke deteriorating changes like aging
found in multicellular organisms and DNA damage may magnify biochemical pathways that
regulate a cells growth or control DNA replication with DNA repair. In the article "DNA Damage,
Homology–Directed Repair, and DNA Methylation" written by Concetta Cuozzo, Antonio
Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may
induce a DNA double–strand break within a genome as well as when DNA methylation is induced
by homologous recombination that it may somewhat mark its reparation through a DNA segment
and protect its cells against any unregulated gene expression that may be followed by DNA damage.
The experiments used to demonstration how gene conversion can modify methylation pattern of
repaired DNA and when that occurs methylation is able to silence the recombined gene. When
exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an
induced double strand break that can be found at a specific location or DNA sequence in where the
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Alzheimer's Disease Research Paper
Alzheimer's disease
Dementia is a broad category of brain diseases that cause long term loss of the ability to think and
reason clearly that is severe enough to affect a person's daily functioning. Alzheimer's disease (AD)
is the most common form of dementia. It primarily affects mainly older adults. Memory impairment
is an essential feature of AD.
Human central nervous system (CNS) is composed of neuron and glial cell ( astrocytes,
oligodendrocytes, microglia) which act as structural support of brain and also contribute for
formation of synapse. Microglia comprises 10% of the adult CNS. When there is tissue injury,
trauma or pathogen, ramified microglia will undergo biochemical changes and turned into an
activated microglia. The cell body ... Show more content on Helpwriting.net ...
The experiment model Include a hypertrophic heart rat (HHR) and a normal heart rat (NHR). The
HHR model showed no cardiac hypertrophy at maturity. Tissue from both HHR and NHR had been
extracted. The cardiac weight index (CWI) was collected. The cardiomyocyte were then obtained
via enzymatic procedure from both neonatal and adult NHR na HHR. Nuclei are then stained.
Polymerase chain reaction (PCR) were used to search mRNA expression levels of ANP,
BNP,MHC,p53, bax–1 and bcl–2 in ventricular tissues of HHR and NHR. Pl3K assay and western
blot analysis were then performed. Lastly, a statistical analysis with value P<0.05 was obtained. The
disadvantages of using this method were that the western blotting method is consider as a time
consuming process that also require high technical demand. However, the interaction between
antibodies and specific target protein can be clearly outlined. This will increase the sensitivity and
specificity in the mixtures that contain mixture of
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Ultrasound Gene Delivery
Ultrasound–based approached can facilitate drug and gene delivery by increasing the transport of
therapeutic agents across the cell membrane and endothelial barrier. The function of ultrasound is
that it can induce cavitation, which is a process of nucleation, growth, and oscillation for gaseous
cavities. The cavitation can cause strong physical–chemical and biological effects in tissues1.
Subsequently, the collapse of cavitation bubbles will disruptive the membrane of cells and tissues to
enhance drug transport. Use the ultrasound to enhance cell permeability is a new methods for gene
transfection. The increase uptake of DNA or RNA has been demonstrated by several investigators2–
1, 2–5. However, the further enhancement of gene transfection ... Show more content on
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So, the combination of ultrasound and transfection agents and Gene encapsulated in the agents could
be a promising way to enhance gene delivery and transfection efficient significantly.
The newly developed acoustically–active echogenic liposome in the form of a liposome associated
with a gas bubble provides a platform for such applications. Liposomes composed of
polyethyleneglycol, DSPC, and perfluoropropane gas provided a 60% increase in expression of
green fluorescence protein plasmid DNA in rat eyes when ultrasound (1.2 W/cm2, 20 s, duty cycle
50%) was applied. The transfection efficiency of such bubble liposomes was higher than that of the
popular lipofection agent, Lipofectamine 2000.
Limitation:
Ultrasound causes perturbation of cell membranes through the interaction of cavitation bubbles with
the cells. This can lead to lysis of cells. Sonoporation appears to be injurious to the cells, thus, the
condition of ultrasound should be careful controlled, to minimize side
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The Role Of Mir 125a And Mir 223
Experimental Design
Aim1. To define the role of miR–125a and miR–223 in modulating Aicda and Prdm1 expression,
CSR and plasma cell differentiation, by overexpression of miR–125a and miR–223 or inhibiting
these microRNA, white mRNA "sponge" that competitively inhibit miR–125a or miR–223 using
retrovirus construct.
Aim1.1 Testing miR–125a overexpression can be performed via transforming a retrovirus vector
construct into E.coli plasmid to amplify the vector, following by isolation of plasmids using specific
Mini–prep Kit. Then, plasmids are transfected into Human Embryonic Kidney 293 cell line
(HEK293 cell) or CH12F3 cell line. To determine the efficiency of transfection, we monitored green
fluorescent protein (GFP) reporter construct expression by conducting either immunofluorescent
assay or fluorescence–activated cell sorting (FACS) 14. We transduce the virus containing miR–
125a vector into primary B cells that is purified by mouse B cell isolation Kit and stimulated
primarily with LPS followed by second stimulation using LPS and IL–4. Cells were collected 60 h
later for qRT–PCR and RNA extraction, or 96 h later for surface Ig analysis by FACS. The
stimulated B cells are stained with CD19 and CD138 to measure the percentages of switched B cells
by FACS. We predict a reduction in IgG1, IgG3, and CD138 populations due to the impede function
in CSR and plasma cell differentiation due to miR–125a expression, which target Aicda and Prdm1.
In addition, RNA extraction will be
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The Rna Interference ( Rnai ) Pathway
The RNA interference (RNAi) pathway is an important biological system that is utilised by many
organisms as a method of regulating gene expression. But what exactly is RNAi? And what is the
importance of its use and application in biology?
RNAi is a cellular process that actively silences specific genes. This method of post–translational
gene regulation has been observed in many organisms including plants, fungi, some bacteria and
animals. RNAi inhibits protein synthesis of the target transcript by the use of small RNA molecules
that target messenger RNA (mRNA) within the cytoplasm following transcription by an RNA
polymerase. RNAi occurs during translation of the mRNA by inhibiting the action of Ribosomes
that catalyse this process within ... Show more content on Helpwriting.net ...
Upon binding the two strands of siRNA are separated, with one strand – known as the guide strand –
remaining bound to RISC and the other strand being degraded. The guide strand then directs RISC
towards its target mRNA for cleavage (Zamore, et al. 2000). This mechanism is extremely precise as
it relies on complimentary base pairing between the bound siRNA and its target mRNA. This
complementarity allows RISC to bind to the target mRNA where the Argonaute protein catalyses it's
cleavage, resulting in degradation of the target mRNA (Fig. 1).
This mechanism inhibits translation of the protein for which the target mRNA codes for and
therefore silences that specific gene. RNAi can also involve the use of coded short hairpin RNAs
(shRNAs), which are larger dsRNA molecules containing a loop section which folds back on itself
to form a double stranded molecule contains a sense and antisense strain. The shRNAs are cleaved
into siRNAs when they bind to RISC and can also found in examples of exogenous introduction
such as viral or experimental insertion. The ability to post translational control of gene expression
with a precise sequence specific manner has been exploited experimentally in researching gene
knockdown and loss of function gene analysis.
In 2006, Andrew Fire and Craig Mello were awarded the Nobel Prize in Physiology or Medicine for
their work in RNAi in Caenorhabditis elegens. Their 1998 paper demonstrated RNAi of endogenous
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Dysbindin-1 Case Study
Dysbindin–1 promotes Drp1 oligomerization
Mitochondrial fission requires dynamin–related protein 1 (Drp1). Drp1 is a large GTPase that can
assemble into oligomers with enhanced GTPase activity. Drp1 is predominantly located in the
cytosol and can be recruited to mitochondria to mediate mitochondrial fission by forming an
oligomeric ring around the mitochondrial tubule to constrict and sever mitochondrial outer
membranes 38–43. Having found that dysbindin–1 regulates mitochondrial fission, we proceeded to
test whether dysbindin–1 is involved in the association of Drp1 with mitochondria. Cultured
hippocampal neurons (DIV14) were transfected with the mitoDsRed plasmid along with the
dysbindin–1 siRNAs, scrambled siRNAs, dysbindin–1C or –1A ... Show more content on
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The mitochondrial fraction of sdy hippocampi contained more monomeric Drp1 and fewer higher–
order Drp1 oligomers than that of WT hippocampi, while the whole cell lysate of WT and sdy
hippocampi had comparable amounts of monomeric and oligomeric Drp1 (Figure 4A–C). These
results indicate that dysbindin–1 is required for Drp1 oligomerization on mitochondria.
Given that dysbindin–1C promotes the recruitment of Drp1 to mitochondria (Figure 3A–C), we
assessed Drp1 oligomers in cultured hippocampal neurons transduced with lentivirus expressing
dysbindin–1C or –1A. Overexpression of dysbindin–1C, but not –1A, increased higher–order Drp1
oligomers and decreased monomeric Drp1 in the mitochondrial fraction (Figure 4D–F). Hence,
dysbindin–1C promotes Drp1 oligomerization on mitochondria.
How does dysbindin–1 regulate Drp1 oligomerization? Dysbindin–1 has been detected on
mitochondrial outer membranes by electron microscopy 28. It is possible that dysbindin–1 binds to
Drp1 to enhance its oligomerization. To test this possibility, we transfected HEK–293 cells with
YFP–Drp1 along with HA–tagged dysbindin–1C or –1A, and used an anti–HA antibody to
immunoprecipitate dysbindin–1 at 2 days after transfection. Drp1 was detected in the
immunoprecipitation product from cells transfected with dysbindin–1C, but not with –1A plasmid
(Figure 4G), indicating that
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Glutamate Transmission
Genetically encoded fluorescent probe for visualizing glutamate neurotransmission in organotypic
hippocampal slices Neurotransmission plays an essential role in communication between neurons. In
such process, neurotransmitters are released by the axon terminal and those bind to a receptor on the
dendrite of another neuron. Among all neurotransmitters, glutamate is the most significant one in
vertebrates. It is used in a large percentage of circuits in the cerebral cortex that support learning and
complex behaviors. Modifications to transmission of glutamate will affect the function of neural
circuits on a large scale in the brain.[1] Glutamate transmission can be studied at the level of
individual dendritic sites. In this case, recent studies ... Show more content on Helpwriting.net ...
I will image these cells and using two–photon glutamate uncaging, individual spine synapses will be
stimulated. This stimulation involves releasing glutamate close to the spine, in which I would expect
to see an increase in fluorescence when glutamate binds to the genetically encoded sensor. Being
able to analyze the change of fluorescence is important to this experiment because the resulting
measurements describe the kinetics of the tested constructs. I have been working on this project
since the summer and we obtained data of these candidate sensors in HEK cells. From this data, I
selected the ones that showed robust signals and tested them in
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Circumventing Traditional Bottlenecks In Glioblastoma...
Circumventing traditional bottlenecks in glioblastoma genetic therapy: ultra–pH sensitive
nanodelivery vehicles RNAi–based modalities offer sequence–specific knockdown of tumor
metabolism, presenting a novel alternative to ineffective treatments. This study investigated a pH
sensitive siRNA nanoparticle which circumvents traditional barriers in genetic therapy by promoting
intracellular delivery, reducing cytotoxic off–target transfections, and facilitating endosomal escape
of siRNA. Acyl–CoA synthetase 5 (ACSL5) was explored as a target for knockdown of glioma
viability. Poly(2–(diisopropylamino) ethyl methacrylate) (PDPA) nanoparticles were synthesized via
nanoprecipitation, measured for cell uptake with fluorescence microscopy, ... Show more content on
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PDPA NP–siACSL5 inhibits glioblastoma proliferation via fatty acid oxidation disruption
siACSL5–PDPA NP transfected U87MG cells and were measured for growth and survival inhibition
via Ki67 staining and fluorescence microscopy. Cells transfected with naked siRNA exhibited 55%
glioma proliferation as compared to the control group which amassed a 78% proliferation rate
(p>0.05), exhibiting the poor efficacy of non–encapsulated siRNA. In contrast, PDPA NP–mediated
inhibition of cell proliferation resulted in less than 22% proliferation and five fold inhibition of
tumor cell proliferation compared to the control group (Fig. 8; p<0.05). siACSL5–PDPA
nanoparticle complexes disrupt the ACSL5–Midkine pathway, leading to activation of downstream
pro–apoptotic proteins such as caspase–3 and simultaneous knockdown of proteins associated with
cell cycle survival such as Midkine, mTOR, and PKB. The two–pronged knockdown of both glioma
proliferation and survivability via forked pathways offers an effective therapeutic solution to combat
tumor viability. Conclusions The PDPA NP formulation serves as an efficient transfection platform
due to its ability to bind to siRNA and condense/stabilize large
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Transfection Lab Report
Transfection of pCas9–GFP with HEK293 cells Introduction Transfection is the insertion of plasmid
DNA into eukaryotic cells. Transfection is used to look at the expression of a gene inserted in a cell.
For this laboratory, the eukaryotic cells used were HEK293 cells. 293 cells are derived from human
embryonic kidney cells. The reagent used to transfect pCas9–GFP into 293 cells was
polyethylenimine (PEI). PEI is a cationic polymer and is the transfecting agent used to introduce
pCas9–GFP into the 293 cells via endocytosis and thus releasing the DNA into the nucleus.
OptiMEM was also used for the transfection of the cells; it provided a biological environment to the
eukaryotic cells, thus increasing the rate of transfection. Following the ... Show more content on
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Next, in figure 2, the 96–well plate shows a change of color after the incubation of BCA reagent and
both dilutions. Wells 9 A–B contained the 1:5 dilution, while wells 9 E–G contained the 1:10
dilution. The BSA standards are found in wells 1, 3, and 5 A–G. Wells 1, 3, and 5 H were used as
blanks; they only contained 40 μl of IP lysis. The results obtained from the spectrophotometer were
used to create the graph and obtain the equation found in figure 3. The equation was used to find the
concentration of the protein, which is 3.45 μg/μl. Figure 4c shows two bands obtained from the
Western blotting performed on GFP; the bands were compared to the protein scale (Figure 4a). The
first band is at ∼25kDa and the second band is at
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Mammalian Cell Culture Lab Answers
Mammalian Cell Culture Lab Manual
Student guide of Cell Biology Laboratory Techniques
2012
Jay M. Feldman
Cell Biology Professor Lagier
11/26/2012
Mammalian Cell Culture Lab Manual
Student guide of Cell Biology Laboratory Techniques
2012
Jay M. Feldman
Cell Biology Professor Lagier
11/26/2012
Table of Contents
I. Introduction * About the author page 2 * Preface page 3
II. Laboratory Techniques * Cell Culture Technique page 4–5 * Sterile Technique page 6–7 * Media
Preparation page 8–9 * Seed cells page 10 * Subculture page 11–12 * Cell count page 13–14 *
Cryopreservation page 15
III. Laboratory Experiments * Intentional Contamination page 16–17 ... Show more content on
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A. They are immortal B. They are epithelial cells C. They are cancer cells D. All of the above
3) What is an advantage of cell culture? A. Control the physiochemical environment of an
experiment B. It is more fun than anti cell culture C. It is easier than anti cell culture D. All of the
above
Answers: 1) C 2) D 3) A
Introduction to Sterile Technique
* What is Sterile Technique?
Sterile technique is the process of executing cell culture procedures without introducing
contaminating microorganisms (bacteria) from the environment
* Sterilize vs Sanitize
The difference between sanitization and sterilization is that when you sanitize something, you are
getting rid of MOST of the live bacteria. It takes about 30 seconds and we perform this using 70%
ethanol or Lysol. When we sterilize something, we make free of ALL live bacteria. Sterilization is
done with an autoclave or by gas sterilize (this is not done in lab).
–Something that is sterilized is also considered sanitized, but if something is sanitized, it is not
sterile.
* Personal Hygiene
By definition, personal hygiene is the act of grooming, cleaning, and looking after our bodies. In cell
biology lab, personal hygiene will be necessary in order to keep contaminants from your body from
reaching your cells. By having good hygiene, you will be able to reduce most of the risk of
spreading bacteria by getting rid of it before you
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Biology Case Study
Materials and methods
The levels of microRNA–22–3p (miR–22–3p) and TP63 were analyzed by RT–qPCR and/or
Western blot. Cell proliferation was measured using MTT assay. Cell invasion and migration were
evaluated using Transwell assay chambers. Prediction of a regulatory relationship between
microRNA–22–3p and 3'–UTR of TP63 mRNA was performed by a bioinformatics algorithm and
confirmed by a dual luciferase reporter assay.
OC cell lines
Lung cancer cell lines H292, PC–9, CL1–5, A549 and normal NHBE cells were used in this study.
All of Lung cancer and normaL ceLL Lines – were provided by ATCC. H292, PC–9, CL1–5, A549
and NHBE cells were cultured in RPMI–1640 medium supplemented with 10% fetal bovine serum
(FBS).
MiRNA and RNA interference ... Show more content on Helpwriting.net ...
The experiments were repeated thrice.
Cell migration and invasion assays
In vitro cell migration and invasion were examined using the Boyden Chamber Assays. For the cell
migration assay, 30000 cells in 100 μl DMEM medium devoid of fetal bovine serum (FBS) and a
given concentration of cetuximab were cultured on a polycarbonate membrane insert coated with
fibronectin in a Boyden Chamber (Essen BioScience, UK). In the lower chamber, 500 μl DMEM
with 10% FBS was added as chemoattractant. After the cells were incubated for 6 h at 37 °C in a 5%
CO2 atmosphere, the insert was rinsed using PBS and cells on the upper side of the insert were
gently removed using a cotton swab. Subsequently, cells that adhered to the lower side were fixed
with methanol, stained with crystal violet solution and counted under a microscope in five
predetermined fields (×200). All assays were independently repeated at least thrice. The same
protocol as described above was used for the cell invasion assay, except that the transwell filter
membranes were pre–coated with 24 μg/μl matrigel (R&D Systems, Inc., Minneapolis, MN, USA)
and the cells were incubated for 8 h at 37 °C in a 5% CO2 atmosphere.
RNA isolation, reverse transcription and RT–qPCR
RNA was extracted from the OC cells using the Trizol reagent. For miR–22–3p, RNA was
transcribed into complementary DNA and amplified using miRNA PrimeScript RT Enzyme Mix kit
according to the manufacturer's instructions
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Essay On Cosisp
I believe that CRISPR is going to, and already is revolutionizing medicine. The way we fight
disease, cure cancer, and maybe even create new humans. The editing techniques I'm about to
mention allow scientist to modify DNA sequences in the human body and.
So what is CRISPR? We first have to understand where CRISPR comes into play, and that is
genomic editing. Genome editing is where engineered nucleases are either inserted, replaced, or
deleted in a living organism's genome, and genomic editing is done with the help of the system
CRISPR. The CRISPR cas system is an RNA guided nuclease system for targeted introduction of
doubled stranded DNA cleavage. It was originally discovered in bacteria as an acquired defense
against foreign ... Show more content on Helpwriting.net ...
Other vector variations are also available to provide added convenience, such as GFP for
transfection monitoring or lenti–backbone for viral delivery. For researchers who perform mRNA
microinjection or mRNA transfection. The applications for CRISPR Cas 9 are endless.
There are two types of editing that are commonly used: homologous recombination and non–
homologous end joining(NHEJ). Homologous recombination utilizes a repair template The desired
changes, such as gene knock out, gene tagging, and specific mutations, are flanked by left and right
homologous arm sequences. Upon double cross over the desired changes integrate into the genome.
In non–homologous end joining the ends are joined together and introduce random mutations. Both
types are used to remedy double strand breaks (DSBs), but homologous recombination is highly
preferred over NHEJ because this type of editing is extremely error prone. It can delete or add
genetic information and cause chromosomal translocations.
So why aren't we using these mechanisms on humans in order to control our genetic makeup? In the
medical field we see great opportunity to treating and even curing some of the most horrible of
disease, yet we are still hesitant. Just as easy, as it will soon be, to edit genes for healing purposes
scientists, researchers, and physicians will have to embrace the uncertainty that comes with it. The
uncertainty of incorrectly placing DNA, or deleting/adding DNA that
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Western Blotting
Second, for the western blotting, the primary antibodies that used to recognize the protein of interest
(FLAG–PTEN) are rabbit anti–PTEN antibodies and the secondary antibodies that bind to the
primary antibodies and convalently linked substrate to produce visible signal are goat anti–rabbit
conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used
to immobilize protein of interest are anti–FLAG monoclonal antibodies from morine cell. As
introduction above, those antibodies come from different species. In order to be successful in
western blotting, the primary antibodies recognize the protein of interest as well as bind to it.
Consequently, rabbit anti–PTEN antibodies were used to bind to FLAG–PTEN. Meanwhile, goat
anti–rabbit conjugated HRP antibodies, the secondary antibodies, ... Show more content on
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As a result, the calculated FLAG–PTEN molecular weight is nearly 72KDa. When comparing the
previous research, the theoretical molecular weight of PTEN is 47KDa, the experimental ones is
25KDa more. The reasons would be [1] since the PTEN was tagged with the FLAG tag and the
FLAG tag itself has its own weight. Although FLAG tag is small in size and contains eight amino
acid, it would affect the finial molecular weigh of FLAG–PTEN; [2] The FLAG–PTEN were
expressed in the mammalian cell system, its original environment. Therefore there would be some
proteins that interact with PTEN naturally in mammalian cell, referring as interacting partner, and
they bind to FLAG–PTEN in the cell lyaste sample then precipitate together in the IP. Those
interacting proteins would increase the molecular weight of FLAG–PTEN by forming a complex
and result in a higher weight band; [3] The resolving ability of the SDS–PAGE is not enough for the
completely separation of the sample, as some of the lower weight markers were compacted in the
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The CRISPR/Cas9 System
Abstract The CRISPR/Cas9 system is an accurate, cheap, and fast way to edit genes. It is used to
edit parts of the genome by removing, adding, or altering some sections of the DNA sequence. The
CRISPR/Cas9 system was used with HIV cell lines and RNA–directed gene editing to prevent new
HIV–1 infections. Both experiments resulted in the stopping of the reactivation of the HIV infection.
HIV (Human Immunodeficiency Virus) puts its own genome directly into a host cell during the time
of infection (Ebina et al, 2013). This can lead to AIDS (acquired immunodeficiency syndrome) if it
is left untreated. AIDS is caused by a retrovirus. HIV can remain dormant for years once its DNA is
in the host cell (Fessenden, 2015). HIV infects ... Show more content on Helpwriting.net ...
By using the CRISPR/Cas9, it is possible to alter DNA to correct genetic diseases in animals and
modify DNA sequences in embryonic stem cells (Rogers, 2016). This opened up the process of
genome modifications in humans through the germ line (sperm and egg) (Rogers, 2016). Doudna
became interested in the CRISPR and found out it is part of the bacterial immune system. It all
started with RNA sequences from invading viruses that become part of the bacterial genomes. From
there the viral sequences stay as DNA in the spacers between the short repeating blocks of bacterial
DNA sequences (Rogers, 2016). When the virus attacks the bacterial cell again, the spacer DNA is
converted to RNA. A Cas9 enzyme and a second RNA molecule attach themselves to the new RNA
and then searches for a matching strand of viral DNA (Rogers, 2016). The Cas9 cuts the viral DNA
which prevents the virus's replication. Charpentier and Doudna found that the guide RNA sequence
could change the direction of the Cas9 to a precise DNA sequence. By this finding, it transformed
the genome engineering and could be treatments for human diseases (Rogers,
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Bb Ap Essay
APPROACH 3
Towards a diagnosis of Bb–Ap co–infection and Lyme neuroborreliosis
Currently, the effective diagnosis of Lyme disease is challenging. Usually, a two–steps serological
test approach is recommended, using ELISA and Western Blot, but a high incidence of false–
negative results is observed. Molecular diagnosis based on PCR are not recommended since Bb
DNA may persist in the host long after the bacterial elimination. Consequently, there is an urgent
need to develop a specific diagnostic tool for Lyme disease, including during Bb–Ap co–infection
and Lyme neuroborreliosis.
3.a. Identifying targets of Bb–Ap co–infection
In order to identify possible diagnostic targets, I will take advantage of the in vivo Microbial
Antigen Discovery (InMAD) approach23. This methodology, especially useful vis–à–vis of
pathogens that are present in very small quantities in the blood, is based on the immunization of
mice using filtered serum from infected animals, in order to promote an immune response against
circulating microbial antigens, but not against cell–related antigens. Then, the immunized sera are
tested against bacterial lysates in 2D–western blots and positive spots are identified by mass
spectrometry as potential diagnostic candidates.
3.b. Developing antibodies against Bb–Ap ... Show more content on Helpwriting.net ...
In the unlikely scenario that this double–transfection might alter viability/virulence of these
microorganisms, I will consider the single–transfection with only bioluminescent genes. The
double–transfection is more challenging and risky, however, it generates completely versatile
microorganisms to be used in a wide range of image–based techniques, consequently, fully
justifying this high–risk/high–reward strategy. Importantly, I used a similar strategy to produce
bioluminescent/fluorescent Trypanosomatids parasites24, underscoring the presumable feasibility of
this experimental
... Get more on HelpWriting.net ...
Human, Eukaryotic And Vitro Associations Of Murine Sec...
Transfection Lab
Abstract: This lab was conducted to determine the efficiency of transfection in HEK293 cells using
cell culture and lipofectamine by growing the HEK293 cells out in growth medium, and then
passaging them into 24 well plate at 50% confluency. After the passaging of the cells, and aspirating
the media after letting the cells grow in the wells for 24 hours, then a master–mix of lipofectamine
and GFP plasmid DNA was added to 500 microliters of stock DMEM, and then adding 100
microliters of the master–mix to each well, and allowing then to grow overnight.Then the next day
accutase was added to dislodge the cells and placed on a side to view them under a UV microscope
to find the efficiency of the transfection. The experimental had the same procedure ... Show more
content on Helpwriting.net ...
Our hypothesis was that there would be efficiency would be lower, and it was supported by the
results obtained in the lab run. The results that we received are in correlation to a paper by Thermo
Fisher, it makes sense because Thermo were the ones we received the procedure and the materials
from to complete this lab. This is because the procedure is optimized for the highest efficiency. The
dependent variable is the efficiency of the transfection of the HEK 293 cells, and the independent
variable is the amount of Lipofectamine 2000 used in the master–mix. The lab gave the result that
the efficiency of the control was 39.6% and the experimental 30.0%, showing a difference in the
efficiencies of the two groups. It was found in a t–test that the results were statistically significant,
the p value is lower than 0.05, and the p–value was 0.0000333530994, which is less than .05. There
were errors in goring out of cells ranging from contamination and the cells not attaching and
growing, there was an error in the first time doing transfection when the instructions were not clear
of how much GFP plasmid was needed in the mastermix, too much was used and there was not
enough plasmid left to fill and the wells with medium. Future implications of this research would be
to grow out more complex cells in culture and
... Get more on HelpWriting.net ...
Human, Eukaryotic And Vitro Associations Of Murine Sec...
Specific Aim 2: To Determine If Gleevec Is Effective In
Specific Aim 2: To determine if Gleevec is effective in inhibiting in vivo cell proliferation in TNBC
cells with overexpressed Arg A recombinant plasmid containing a viral promoter driving Arg
expression will be inserted into TNBC cells. Another empty recombinant plasmid will be inserted
into different TNBC cells. Another plasmid containing a promoter driving Arg expression will be
inserted into another group of TNBC cells. Soon after, an immunoblot will be run using these three
different TNBC groups' cells to ensure the proper transfer of plasmid. The proteins we will test for
include Arg, Abl, actin, and Ras–MAPK. Actin is again serving as a positive control, while the
expression of Ras–MAPK proteins can act as a quantitative ... Show more content on
Helpwriting.net ...
Abl is known to mutate its kinase domain so that Gleevec cannot effectively inhibit it and cellular
proliferation (Lovera et al., 2015). A western blot will be run to determine the extent this mutation
will affect our experiment at the beginning and 4 months into our experiment. Some tumors from all
three cell lines will have their cells lysed and run through the gel, which will test for both Abl and
Abl–Gleevec compound, as well as a major substrate for Arg–Abl, ArgBP2.
Results–
The western blot containing the three different cell lines expressed an equal amount of actin
expressed in all three lanes. This served as a control to ensure the plasmids were inserted correctly
and there was no interference of any other gene. The lane with cells from mice group 1 showed
darker bands on lines corresponding to the proteins Arg as opposed to the lane with mice group 2
cells. The bands corresponding to the protein Abl appeared to be the same on both lanes,
demonstrating that Abl was not being accidentally overexpressed as well. These pieces of
information elucidated to us that the Arg gene was being successfully overexpressed in line 1. The
bands corresponding to Ras–MAPK were darker for mice group 1 than for group 2, demonstrating
that this signal transduction pathway for cell proliferation has been downregulated with the
overexpression Arg.
... Get more on HelpWriting.net ...
Human, Eukaryotic And Vitro Associations Of Murine Sec...
T Cell Gene Therapy : The Center Of Research With Stem...
The CCR5 co–receptor is not only the center of research with stem cell transplant, but has also been
a recent target of gene therapy research. Gene therapy is a fairly new technology where genes
(edited or normal) are transplanted in humans to produce a specific response. One avenue of
research that is currently being studied is T cell gene editing focused on the CCR5 delta32 mutation
in HIV infected patients using the CRISPR/cas9 system. There is some research using TALENS, but
evidence with CRISPR/cas9 will be discussed here (Ye, 2014). The CRISPR technique that edits
gene sequences has been also researched for use in Hepatitis B, Epstein Bar Virus, Malaria, and
Human Papilloma Virus. Simply put, the CRISPR/cas9 system can be ... Show more content on
Helpwriting.net ...
The researchers created and studied the best way for the CRISPR/cas9 system to be delivered into
CD4 cells. The best delivery mode for the CRISPR/cas9 was assessed to be an adenovirus known as
the Ad5F35, and the optimal time for exposure for maximal gene editing was found to be 8 days.
The CRISPR/cas9 was introduced to the cells by the Ad5F35 adenovirus and eight days passed to
allow the system to modify the genetic makeup. Following transduction of the CD4 cells with the
genetically engineered CCR5 mutation, the cells remained HIV resistant. This study claims to be the
first to successfully use an adenovirus to transduce genetically modified DNA into CD4 cells to
provide resistance to HIV. In theory, these modified CD4 cells can then be transplanted in HIV
patients so that their fighter cells will be resistant to HIV infection. (Li, 2015).
Another recent study involved transplanting these modified T cells into mice using the CRISPR/cas9
to see if the engineered cells would transfer resistance to HIV. After transplanting the T cells and
exposing the mice to HIV, the mice tested resistant to the virus (Zu, 2017).
The author in the initial CRISPR study, Li, believed it to be important to create a way to genetically
change the CXCR4 co–receptor as well as the CCR5 co–receptor so that the virus cannot utilize any
receptor to enter the host cell (Li, 2015). In a study done in 2017, scientists developed a way to alter
the genetic makeup of the CXCR4 co–receptor. Instead of
... Get more on HelpWriting.net ...
Human, Eukaryotic And Vitro Associations Of Murine Sec...
Transgenic Mice Essay
Transgenic Mice, Care and Handling Double transgenic mice express a chimeric mouse/human
amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1–dE9) were
purchased from The Jackson Laboratory (004462, Bar Harbor, ME, USA). All animal treatments
were approved by the Animal Ethics Committee of Tsinghua University. Mice had access to pre–
filtered sterile water and standard mouse chow and were housed under a reversed day–night rhythm
in accordance to local legislation on animal welfare. Cell Lines and Reagents A172 and U251 cell
lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and the
Chinese Academy of Sciences (Kunming, Yunan, China), respectively. Cell cultures were
maintained in ... Show more content on Helpwriting.net ...
Transfection and selection was pursuant to previous description [40]. The empty vector served as the
negative control. For the EGFP–LC3 cell line, the pEGFP–LC3 overexpression plasmid was
previously prepared in our lab and used for cell transfection, followed by G418 (100 μg/ml)
selection. Transient Transfection Cells were transfected with the miR34a, an antisense miR34a
inhibitor (anti–miR34a), siSH3GL3 (5′–GUAAUGCAUUGAUAGAAGU–3′), siCTSB (5′–
AGAGAGUUAUGUUUACCGA–3′), siSH3GL2 (5′–GAACCAAGCUAGAUGAUGA–3′),
siSH3GL1 (5′–GCAAGGCGGUGACAGAAGU–3′), and siPPP1R18 (5′–
GCUCGAGAAUGGACACCCA–3′), using Lipofectamine LTX with Plus Reagent (Thermo Fisher
Scientific) according to the manufacture's recommendation. A scramble siRNA (5′–
UUCUCCGAACGUGUCACGUTT–3′) was used as negative control. 48 or 72 h after transfection,
cells were treated as indicated. Western Blot (WB) Cells were lysed with Laemmli buffer, and each
lysate sample was loaded into a polyacrylamide gel and separated by electrophoresis at 30 mA
following by transfer onto PVDF membranes (Millipore). Membranes were blocked for 1 h at room
temperature using 5 % skim milk in Tris–Buffered Saline with 0.1 % Tween–20 (TBST). WB
analysis was
... Get more on HelpWriting.net ...

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Human, Eukaryotic And Vitro Associations Of Murine Sec...

  • 1. Human, Eukaryotic And Vitro Associations Of Murine Sec... INTRODUCTION In eukaryotes, Usually the UGA acts as a stop codon in transcription but the elongation factor, known as SELB, has a C–terminal SECIS RNA binding domain and N–terminal Sec–tRNA domain, inserts the selenocystiene at the stop codon. An SECIS binding protein, known as SBP2 is found in mammals but it lacks EF functionality. We will describe the in vivo and in vitro associations of murine SEC–specific EF (eEFSec) element with SBP2 and SECIS in order to correlate their function in selenoprotein synthesis. RESULTS Extensive database search of EST revealed candidates for human, eukaryotic and murine Sec– specific EF. Murine EST indicates a 583 amino acid protein while the eEF1A shows 462 amino acids. The protein is homologous to EF and also has portions of eEFsec family. Similar to SELB, eEFsec lack insertions that are present in eEF1A, responsible to GTP exchange factors interaction. Homology of c terminal extends both to eukaryote and archeal proteins. Murine tRNA binding, assessed by nitrocellulose filter binding assay showed high binding with the two isoforms of Sec–tRNA and lower levels of binding with seryl–tRNA, methionyl–tRNA etc. A Kd of 0.11 μM was found by scatchard analysis for GTP binding, 3 times less than for GDP binding, showing that GTP exchange factor is not needed. These results show Sec–tRNA–specific EF. Co–immunoprecipitation studies were performed to check the hypothesis that binding of SBP2 to SECIS resulted in recruitment of eEFsec–tRNA. We ... Get more on HelpWriting.net ...
  • 3. Biotherapeutic Therapy Biotherapeutic (DNA and Nucleic acid delivery) Also, biotherapeutic therapy is similar to chemotherapy except that instead of delivering small molecular drugs (or metabolite ... etc.), biological agents such as DNA, small interfering RNA (siRNA), proteins, and peptides are delivered to tumor sites to induce apoptosis and cell death. In cancer, damaged DNA results in defect in protein expression causing deleterious effects. Cancer may be treated with DNA delivery by replacing the defective genes within cancer cells. Alternatively, cancer therapy via siRNA works by suppressing the protein expression of damaged genes. Protein and peptide therapy, on the other hand, operates by attacking specific cell mechanisms such as disrupting cell adhesion, interfering with angiogenesis, and/or blocking other ... Show more content on Helpwriting.net ... In the past, delivery of biotherapeutics has shown limited success due to the immunogenicity caused by the delivery vectors [154] (were the scientist were using the virus as a vector). Inclusion of biotherapeutics in NP delivery systems has promise as biocompatible polymer–coated NPs provide protection against inhibitive immune responses and targeted delivery of these therapeutic agents. Typically, for gene therapy, NPs are coated with a cationic polymer that encapsulates or binds DNA or siRNA as the positive charge of this layer allows for favorable electrostatic interactions with negatively charged nucleic acids [155]. The conjugating of NPs with peptides is other design considerations for DNA delivery to the cell nucleus for the successful transfection of genetic material. This is usually accomplished by conjugating peptides that ... Get more on HelpWriting.net ...
  • 5. Binary Chromatography Lab Report Afterward, PTEN purified using affinity chromatography. Since PTEN fused with His6 tag, Ni–NTA affinity chromatography would work to purify PTEN. Ni–NTA is a type of immobilized metal affinity chromatography that contain coupled Ni2+ nickel chelate forming ligand (imiondiacetic acid) that immobilized to agarose. The science behind using Ni–NTA affinity chromatography is that histidine bind specifically and reversibly to the nickel chalet thus washing step will remove other contaminate and then His–PTEN eluted using imidazole. Bradford assay and SDS–PAGE performed to quantify and analyze the progress toward PTEN purification. The calculated concentration of the samples presented in table 8. The Concentration of samples make since having the ... Show more content on Helpwriting.net ... A scientist need to demonstrate mammalian cell transfection under very sterile technique since they grow slowly and affected by contamination resulting in lower expression. In addition, the total number of mammalian cell should maintain steady and once there is confluent, cells have no more room to grow, mammalian cell culture should split using digestive enzyme and placed into new fresh media. To do so, hemocytometer used to count mammalian cell and demonstrate the cell concentration. Table 11 shows the calculation of mammalian cell concentration and for transfecting 1000,000 cell we need 1.17 ml. Following mammalian transfection, immunoprecipitation processed for FLAG–PTEN gained from mammalian cells. Anti–FLAG antibody used as ligand just as affinity chromatography to purify FLAG–PTEN. Latter on, western blotting carried out by transferring the protein from the completed SDS–PAGE to PVDF membrane as the order in figure 5. After that, incubation with primary and secondary antibody to visualize PTEN through the chemiliuminescence reaction as shown in figure 7. The figure present FLAG–PTEN in three samples the FLAG–PTEN IP, supernatant FLAG–PTEN after IP, and lysate FLAG–PTEN before IP. Lysate FLAG–PTEN before IP and FLAG–PTEN IP spouse to contain PTEN and that confirm the success of mammalian transfection. However, the FLAG–PTEN IP showed a smeared band. That may due to high protein concentration load ... Get more on HelpWriting.net ...
  • 7. Essay On Proteasome Recent findings have focused on developing effective proteasome inhibitors as anti–proliferative agents because previous reports strongly suggest that abnormal or impaired functions of proteasome aggravate the accumulation of aberrant proteins in cells, which consequently stimulate apoptosis in cells (Chhangani et al., 2013; Chhangani and Mishra, 2013; Huang and Figueiredo–Pereira, 2010). Although, the role of proteasome is well–established in clearance of misfolded aggregation of proteins linked with neurodegeneration and aging,still critical examination of previous studies suggest that inhibition of proteasome activities can be auseful therapeutic strategy to induce apoptosis in cancer cells, due to their high metabolic demands and ... Show more content on Helpwriting.net ... Mitochondrial Membrane Potential Detection JC–1 Kit (BDTMMitoScreen) and FITC Annexin–V– Apoptosis Detection Kit I (BD PharmingenTM) were obtained from BD Biosciences. Lipofectamine® 2000, OptiMEM, and JC–1 (5,5', 6,6'– tetrachloro– 1,1',3,3'tetraethylbenzimidazolocarbocyanine iodide) were purchased from Life Technologies and Molecular Probes respectively. TUNEL assay kit, Dual luciferase reporter gene assay kit and Proteasome–GloTM assay reagents were obtained from Promega. Anti–ubiquitin, anti–GFP, anti– 20S proteasome, anti–p53, anti–p27, anti–p21, anti–Bax, anti–IkB–α, anti–β–actin and anti–HA antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase–conjugated anti–rabbit–IgG and anti–mouse IgG; anti–rabbit, and anti–mouse (IgG fluorescein isothiocyanate and IgG–rhodamine) were obtained from Vector Laboratories. Plasmid pcDNA™ 3.1 was purchased from Life Technologies. pcDNA3–EGFP (Addgene 13031), pRK5–HA–Ubiquitin–WT (Addgene plasmid 17608), pEGFP–Hsp70 (Addgene plasmid 15215) and Luciferase–pcDNA3 (Addgene plasmid 18964) plasmids were purchased from Addgene. 2.2. Cell Culture, Transfection, Reporter Gene And Cell Viability Assays COS–7 and A549 cell lines were maintained at 37°C, 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) streptomycin/penicillin (Gibco, Invitrogen). For various transfection and treatment experiments, cells ... Get more on HelpWriting.net ...
  • 9. Dendrimers Research Paper Dendrimers Dendrimers are 3D polymers with spherical, highly branched structures. Polyesters, polyamines, and polyamides are commonly used; however, polyamidoamine (PAMAM) dendrimers are the most frequently used due to their high transfection efficiency. The transfection efficiency is highly influenced by the size of dendrimers and the type of amine groups that they are composed of. The primary amine groups participate in DNA binding, so they promote DNA cellular uptake. However, the tertiary amino groups reinforce the release of DNA into the cytoplasm because they act as a proto–sponge in endosomes. Transfection efficiency can be enhanced by increasing the size and diameter of the dendrimers. One example of dendrimer DNA complexes is nitrogen–core ... Get more on HelpWriting.net ...
  • 11. Endoplasmic Reticulum ( Er ) Stress Response Abstract Endoplasmic reticulum (ER) stress response may be mediated by HRV16 nonstructural proteins 2B, which share similar characteristics to CVB3 2B. To understand whether HRV16 2B induces an ER stress response, a HRV16 2B fusion protein with GFP were generated and its effects on ATF6a (activating transcription factor 6a), IRE1 (Inositol–requiring enzyme 1), and PERK (PKR–like ER protein kinas) expression in BHK cells were determined. The results showed that HRV16 2B induced significantly high expression of glucose–regulated protein 78, and activated ATF4 and ATF6 in a time–dependent manner. HRV16 2B also significantly activated the PERK–eIF2α pathway with marked increased in expression of p–PERK, p–eIF2α, and CHOP. Additionally, HRV16 2B decreased the expression of p–IRE1 and ATF6 p50. These results demonstrated for the first time that the PERK–eIF2α signaling pathway, but not IRE1 and ATF6, mediates ER stress in BHK cells. Keywords: HRV16, 2B protein, Endoplasmic reticulum stress, PERK Introduction Human rhinovirus (HRV) infections are major risk factors for asthma and chronic obstructive pulmonary disease (COPD) [1–4]. The airway epithelial cell is the dominant site of HRV infections in vivo [5,6], and both upper and lower airway epithelial cells are infected at a similar frequency [7]. Non–enveloped rhinovirus belonging to the Picornaviridae family is spherical in shape, with a diameter ranging from 28 nm to 30 nm. The viral genome contains a single open reading ... Get more on HelpWriting.net ...
  • 13. PEI Transfection Paper Our project revolved around two major ideas, the first idea that detected the expression of the Hemagglutinin glycoprotein of Measles virus (MV–H) on the surface of helper virus (Vesicular Stomatitis Virus) in our tissue culture. The second idea that identified the effect of Axl antibodies on the penetration processes of VSV–ΔG–MV–H virus The manner of this project represented in three major stages, the first stage that generated the VSV–ΔG–MV–H pseudovirion. This stage required to remove the VSV G glycoprotein gene from VSV and replace with gene for the green fluorescent in helper virus, however, this process was more complex so we skipped this step. The next step that was extraction the plasmid from the bacteria by following a specific protocol and then started working with PEI Transfection. ... Show more content on Helpwriting.net ... For PEI Transfection, we added 1 ml of Trypsin to a T–25 flask of 293T cells and incubate at 37 0C for 10 minutes. Then, we supplemented 10 ml of DMEM (2.5%), FBS (15), P.S medium to the flack and finally transferred to dish and put back in CO2 incubator. After 5 days, we diluted plasmid DNA (ug) in 250 ul of 150mm NaCl in a sterile tube, and diluted 40 ul of PEI into 250 ul of 150 mm NaCl in second sterile tube. After that, incubated each separate tube for 5 minutes at RT and then mixed the DNA tube with the PEI tube. We put the tube in incubator for 2–5 minutes at RT, supplemented dropwise to cells, and put back into incubator. The important steps that was added helper virus (VSV) to cells and put it in the CO2 incubator; then, collected supernatants that contains our ... Get more on HelpWriting.net ...
  • 15. Mir34a Essay MiR34a inhibits Aβ fibril uptake and autophagy flux Regarding the compromised capability of Aβ clearance in AD, we suspected that miR34a may be an indispensable player. To validate this, Aβ42 fibrils were prepared from Aβ42 monomer ex vivo and used to treat glioma A172 and U251 cells. Aβ uptake was blocked by both transient transfection of a miR34a mimic and stable miR34a overexpression (Supplementary Fig. 1B, Fig. 1A), but enhanced by anti–miR34a (Fig. 1B), indicating that miR34a suppressed Aβ phagocytosis in these cells. The fact that enhanced and decreased levels of miR34a correlated with corresponding changes in intracellular Aβ supports the notion that miR34a may slow down Aβ clearance by affecting its Aβ uptake and degradation. ... Show more content on Helpwriting.net ... Since mature autophagosome fuse with lysosomes to become autolysosome in the very late stage of autophagy, we asked whether miR34a could retard autophagosome–lysosome fusion by examining the co–localization of EGFP–LC3 and LysoTracker Red, a specific dye for live cell lysosome labeling [47]. As expected, EGFP–LC3 puncta were dramatically reinforced in miR34a mimic transfected cells but did not co–localize with LysoTracker Red. This was acting similar to cells treated with bafilomycin A1 (Baf), which blocked autophagosome–lysosome fusion. In contrast, E64D treatment induced a remarkable increased EGFP–LC3 puncta, which were well co–localized with LysoTracker Red (Fig. 1D). Since lysosome–associated membrane glycoprotein 2 (LAMP2) is also critical for autophagosome–lysosome fusion [48], we examined the effect of miR34a on LAMP2 expression using WB analysis. MiR34a caused an unexpected increase of LAMP2 (Supplementary Fig. 1C), suggesting that the mechanism by which miR34a blocked autophagosome–lysosome fusion was not due to LAMP2. As LAMP2 is mostly important for chaperone mediated autophagy, its increase might be a compensation for the decrease in macroautophagy. In summary, the above results reinforce that miR34a compromised Aβ degradation by, at least, blocking autolysosome formation. ... Get more on HelpWriting.net ...
  • 17. Rhyme Case Study 3.2.1. Creation of the reporter plasmid with VDREs WT and mutant VDREs fused to a minimal promoter were ordered as premade G blocks with restriction sites to allow cloning upstream of Firefly luciferase (Method xxxx). 3.2.2. Sequential digest of SacI and BglII As there is no buffer in which both SacI and BglII exhibit >50% activity, a sequential digest was necessary (Method 2.3.). The digest was successful and the resulting cells were then lysed. 3.2.3. Transformation using DH5–alpha cells Transformation was carried as instructed in Methods2.4. The cloned VDR was placed in front of the Firefly and Renilla luciferases. A microplate was set up for the cell transfection and the cells were transfected into HEK293T cells (Method2.5.). A dual luciferase assay was then run and luciferase ... Show more content on Helpwriting.net ... This WT stimulation was expected. The difference seen between WT1 and WT2 is due to the use of two different clones being used. The mutants were found to not be acting as expected. However, the mutant UGG is higher than the WT UGG. It is possible that is as a result of the mutant having not been stimulated by calcitriol. Because of this, a fold stimulation plot was produced (Figure 6b). Also with the UGG mutant codon, it can be seen that the calcitriol is not responsive when transcription does not stop at the first stop codon. A final Western Blot was done to analyse the transfected cells. 24 tubes were arranged, each receiving 2.5 μl from each well on the transfection plate to give a total volume of 10 μl of each example because each transfection has four samples available. Two separate polyacrylamide gels were run at 100V for 60 mins. The gels were transferred to a nitrocellulose membrane and treated with primary and secondary antibodies (Methods2.6.). This was followed with three 5 minute washes with PBS–T, and scanned. The results can be seen in figure 7 as ... Get more on HelpWriting.net ...
  • 19. Western Blotting Of Camkii Œí And T 287 Western blotting of CaMKIIβ and T–287 phosphorylated CaMKIIβ Western blotting was performed as described previously (Chapter 3). In short, Proteins were extracted from OLs using RIPA buffer with protease and phosphatase inhibitors. The concentration of extracted proteins was assessed using the BCA assay. Target proteins were probed with primary antibodies for CaMKIIβ (1:1000, Life Technologies) and phosphor–CaMKIIβ (T287) (1:1000, Abcam) at 4°C overnight. Membranes were incubated with appropriate IRDye secondary antibodies (1:3000, Li–COR, Lincoln, NE) for 1 h at room temperature, and imaged using an Odyssey Imager (Li–COR). Protein bands were quantified using Li–COR image studio software. RT–PCR analysis of CaMKII transcription RNAs were extracted from OLs or cerebral cortex of P2 ICR mice using the miRNAeasy Mini Kit (QIAGEN Inc. Valencia, CA). Concentration of RNAs from each sample was determined using NanoDrop ND–1000 spectrophotometer (Thermo Scientific). Equal amount of RNAs were then used as template to transcribe cDNA using a High capacity cDNA reverse transcription kit (Life Technologies). Gene–specific primer pairs were designed using NCBI Gene database and primer design web tool (http://www.ncbi.nlm.nih.gov). The web tool generates a series of primer sets that fit the parameters defined by users. These parameters include size of the final product and the primers, whether primers span the junctions between exons, GC contents of the primer etc. For each isoform ... Get more on HelpWriting.net ...
  • 21. Lab Report Results MiR–365b–3p regulates proliferation, migration, invasion and apoptosis of human lung cancer cells In order to investigate the expression of miR–365b–3p in normal cells and lung cancer cells, we used real–time PCR to measure the expression of miR–365b–3p in various cells. Results showed that the expression of miR–365b–3p was significantly lower in lung cancer cells compared with that in normal cells (Figure 1). After transfection, the expression of miR–365b–3p in COLO 668 cell line was successfully upregulated by miR–365b–3p mimic and inhibited by miR–365b–3p inhibitor (Figure 2A). Cell viability assay showed that miR–365b–3p suppressed cell viability while miR–365b–3p inhibitor promoted cell proliferation (Figure 2B). Furtherly, ... Show more content on Helpwriting.net ... MiR–365b–3p exerts anticancer effect by targeting RAB1B in human lung cancer cells RAB1B siRNA was used to downregulated the expression of RAB1B. Figure 4A showed that after inducing with siRNA, the downregulation of RAB1B was confirmed by western blot. According to our cell viability assay result, transfection with miR–365b–3p inhibited lung cancer cells proliferation, which was promoted by RAB1B siRNA (Figure 4B). Moreover, siRNA and miR–365b–3p were both capable of inducing COLO 668 cells apoptosis by activating caspase 3. And according to our results (Figure 4C, 4D), although there was no statistical significance, the apoptosis–promoting effect seemed stronger in miR–365b–3p group. We also compared the anti–tumor metastasis effects between RAB1B siRNA and miR–365b–3p, the results showed both of them significantly reduced the migration and invasion of COLO 668 cells (Figure 4E, 4F). By comparing with RAB1B siRNA, we furtherly confirmed the miR–365b–3p exerted anti–tumor effect by targeting RAB1B in human lung cancer cells. Discussion Various miRNAs are reported to play roles in lung and other cancer. Based on previous studies, let–7, miR–15, miR–26, miR–29, miR–30, miR–126 and miR–142, have been reported to have abnormal expression in lung cancer cells compared with normal cells [18]. In this report, we first time provides evidence that miR–365 exerts anti–cancer ... Get more on HelpWriting.net ...
  • 23. How Information Is Processed Through The Brain, Especially... The authors in this article are trying to better understand how information is processed in the brain, especially the diseased brain. In order to research this information there first needs to be a precise method of spatio–temporal recording of electrical activity in larger neuronal circuits and individual neurons. The authors hypothesized that fluorescent indicators would be useful tools in recording brain activity because they would allow monitoring of a genetically defined neuronal circuit and they would not require chemical access to visualize and provide optical recording of brain activity. Since the current techniques could not meet the authors' requirements to study neuronal activity in vivo, they sought out to create a voltage ... Show more content on Helpwriting.net ... Next the authors made a circularly altered Clover GFP by creating termini at the amino acid positions 145 and 144 and linking its N– and C– termini using the peptide 'GGTGGS' between the C–terminal (K239) lysine and the original starting methionine. The authors obtained cpsfGFP– OPT145–144 (abbreviated as cpsfGFP–OPT), which is a circular permutant of a superfolder GFP variant and which contains 7 mutations that are not present in the original GFP superfolder; it was made for the use in a split GFP system. The authors then constructed circularly altered superecliptic pHlurinA227D (cpsepHluorinA227D), creating new termini at the amino acid positions 145 and linking its original N–and C– termini using the peptide GTGGSAS between the C–terminal lysine (K239) and a lysine originally near its N terminus (K4). Then authors constructed ASAP1 using the voltage–sensing domain (VSD) from the Gallus gallus voltage–sensitive phosphatase. The ASAP1 variants were made with the alternate VSDs by inserting cspfGFP–OPT following amino acid G146 (Xenopus laevis), G147 (Danio rerio), or amino acids L203 to G214 (Ciona intestinalis). The authors isolated the VSD from the VSP by cutting the C–termiminus at the same positions: T183 (G. gallus), T182 (D. rerio) or S244 (C. intestinalis). Then ASAP1 variants and Archlight Q239 were cloned between the NheI–HindIII sites of pcDNA3.1/Puro–CAG. The scientists used coexpressed sensors with a ... Get more on HelpWriting.net ...
  • 25. Scientific Discovery Of Selective Breeding Essay Introduction For almost 10 milleniums, with "treatises as much as 2000 years old" giving advice on selective breeding, humans have carried out genetic manipulation on other organisms for their own benefit. Humans manipulate the genetics and genetic frequencies of both individual organisms and entire populations in order to raise the frequency of existing or transgenic traits we find useful or desirable. Human manipulation occurs for a large number of reasons, including; increasing profit/productivity in agriculture and farming, creating medical aids for human diseases, increasing survival chances of the given species and opening pathways for scientific discovery. Two major methods humans manipulation can be noted; selective breeding, which is the breeding of organisms within the same species to increase the frequency of existing genes, and transgenesis, the transfer of genes into the genome of one organism to create new traits, are used. Selective Breeding Selective breeding is the process in which humans sexually breed animals or plants for traits that are considered "desirable" for the goal of producing offspring and hopefully; entire lineages, with same such "desirable trait". Techniques of selective breeding includes marker assisted selection; screening a genome for specific genes of the parents before actually breeding them, and the original inbreeding/line breeding which involves breeding organisms with the desired traits with close relatives to maintain the similar ... Get more on HelpWriting.net ...
  • 27. Hiv And The Human Immunodeficiency Virus Essay . Introduction HIV (Human Immunodeficiency Virus) is able to integrate its genome into the host cell genome during infection (Ebina, Misawa, Kanemura, Koyanagi, 2013). This can lead to AIDS (acquired immunodeficiency syndrome) if it is left untreated. AIDS is caused by a very efficient and sneaky retrovirus. HIV can remain dormant for years once its DNA is in the genome of the host cells. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Cas9 was the first nuclease discovered. The focus of the CRISPR/Cas9 is to target a particular disease causing gene and remove it. II. CRISPR/Cas9 with HIV cell lines HIV infects CD4 + T cells. The viral particles of HIV keep their genetic information stored as double–stranded RNA. They use a reverse transcriptase that converts their genome into double–stranded DNA (Edina, Misawa, Kanemura, Koyanagi, 2013). During this process the virus can mutate quickly because many errors occur in the HIV genome. "The DNA is inserted into the host cell genome by integrases which use LTR (long terminal repeat) sequences on the viral genome to integrate with the host DNA" (Ebina, Misawa, Kanemura, Koyanagi, 2013). This results in a provirus that can continuously produce new viruses that infect other immune cells. Doctors can mix different antiretroviral drugs to help with HIV, but once you stop the treatment the virus ... Get more on HelpWriting.net ...
  • 29. Accelerated Sensor Of Action Potentials Accelerated Sensor of Action Potentials 1 The authors were trying to achieve a goal that they describe as a major goal in the field of neuroscience. They were looking for some method of reporting electrical activity in neuronal populations, specifically optical reporting. They developed a voltage sensor that they named Accelerated Sensor of Action Potentials 1 or ASAP1. This allows for GFP to be inserted into an extracellular loop of a voltage–sensing domain (VSD) and making fluorescence responsive to membrane potentials. They were looking for ASAP1 to detect a broad spectrum of membrane potentials, ranging from subthreshold to rapid trains of action potentials. For this to be the case they had to have the appropriate brightness, dynamic range and kinetics. They hypothesized that inserting GFP into the S3–S4 loop would allow voltage–induced movements to interrupt GFP fluorescence. They also hypothesized that a VSD with a shorter loop would increase the coupling between the voltage–induced movements and GFP barrel distortions. They performed various tests and concluded that a particular variant of the GFP improved brightness, dynamic range and expression at the membrane, giving rise to the protein they named ASAP1. From here they performed tests to show that the VSD that they chose initially was the best choice. There are many methods that were used in the achieving of this goal. Plasmids are small circular sections of double stranded DNA that are distinct from a cells ... Get more on HelpWriting.net ...
  • 31. Methods Of Marker Assisted Selection Marker assisted selection (MAS) is a method using fluoresces to determine whether there are mutations in a particular gene. One form of MAS is Fluorescence in situ Hybridization (FISH), in which the target gene (ADA gene) is denatured (splits into single strands) in a solution containing a direct DNA probe which has an identical base pair sequence to the ADA gene being analyzed (these DNA probes are also denatured into single strands). A direct DNA probe is made up of modified dNTPs which have been altered to contain a fluorophore (a fluorescent chemical compound that has the ability to re–emit light upon mild excitation), these will bind to the ADA gene strands if there are no mutations present (i.e. it is a fully functioning gene). If ... Show more content on Helpwriting.net ... In this time the blastocyst also connects to the endometrial blood vessels in order for it to gain the nutrients necessary for survival through the blood supply, it is after this that implantation is considered complete. Medication can also be taken to increase the chances of successful implantation, such as progesterone, progestins or a GnRH agonist, however these are nonessential for successful implantation to occur. Implantation is not 100% successful hence the use of these medications for increased chances perhaps if only a few viable embryos are able to be implanted Gene Therapy Gene therapy is a way in which an individual with a preexisting genetic disorder (in this case ADA deficiency) can be treated. This involves the insertion of a functioning ADA gene into an individual's cells through the use of viral vectors, to replace the mutated ADA gene that is non– functioning or functioning incorrectly. In order for this process to be viable multiple techniques are used, including: restriction enzymes, ligation, PCR and transfection. Through these processes an individual is able to As there are 70 known mutations on the ADA gene currently that can cause ADA deficiency, I have chosen to focus on one specific mutation's gene therapy. This mutation is a substitution mutation located at the 646th base pair on exon 7, where a G base has been substituted for an A base. In this case a fully functioning ADA gene is sourced from a donor, in which it will ... Get more on HelpWriting.net ...
  • 33. Modern Neuroscience, Accurate And Accurate Optical Reporting In modern neuroscience, accurate optical reporting of electrical activity in defined neuronal populations can be monumental task. The reason for the enormity of this task is due largely in part to the lack of a sufficient, effective and accurate optical reporting procedure for activity of the brain. At this point in time, genetically encoded fluorescent indicators seem to be the answer for accurate and effective optical reporting, largely because they allow monitoring of defined neuronal circuits and do not require any chemical access. Ideally, these indicators would produce large fluorescent responses, allowing imaging on how input signals are processed into output signals by neurons. In addition, these ideal indicators would have ... Show more content on Helpwriting.net ... ASAP1 was developed by inserting a circularly permuted green fluorescent protein into a voltage sensing extracellular domain. It's believed that this design will have sufficient brightness, faster kinetics, as well as a dynamic range of detection of neuronal activity needed to make this ASAP1 design the ideal fluorescent based reporter for optical reporting of brain activity. This hypothesis was tested extensively in a laboratory setting to prove the claim. Numerous standard laboratory techniques and instrumentation was used to obtain the forthcoming results. The ASAP1 expression plasmids were created by standard molecular biology methods, and then verified through cloned fragment sequencing. A plasmid was used for multiple reasons, the most prominent being that plasmids are very easy to manipulate to express a desired genetic element. In addition, they are very stable as well as self–replicating once initially constructed. HEK293A cells were used in experimentation and transfected with the ASAP1 plasmid. The HEK293A cells, which are very popular for transfection, were chosen as they are easy to reproduce, maintain, and have a high efficiency of transfection. HEK293A cells also have minimal processes that need to be accomplished in order to preform voltage–clamp experimentation. Transfection is simply the process by which ... Get more on HelpWriting.net ...
  • 35. Gene Therapy Safety Efficacy and Safety of Gene Therapy Zack Martin What is Gene Therapy? A new form of medicine with the potential to save and improve millions of live is gene therapy, or gene replacement therapy. Gene therapy is the use of DNA as a pharmaceutical agent used to treat or cure diseases or disorders caused by defective or missing genes. There are three methods of gene therapy: replacing a mutated gene with a healthy copy of the same gene, inactivating a gene that is not functioning properly, or introducing a completely new gene to help the patient fight a disease. Gene therapy is a promising treatment option, however, the technique has yet to be perfected and is still dangerous and experimental. Replacing a Mutated Gene with a ... Show more content on Helpwriting.net ... The first method is to attach the gene to a virus. This is done by removing the functional group of the virus that harms the body and attaching the gene that is to be delivered into the cell. Retroviruses and adenoviruses are the two types of viruses used in gene therapy. Retroviral vectors are used because it inserts its own DNA into the host's DNA. There are two ways to use retroviral vectors in gene therapy, introduce the vector intravenously, or removing the patient's tissue, inserting the vector in a lab setting, then replacing the tissue. One of the flaws using the retroviral vector method in gene therapy is that integrase enzyme(responsible for integrating the retroviral DNA into the host cell) can insert the gene in any position in the genome. If the gene is place in the middle of another functional sequence, it would cause a malfunction of that particular gene. Not all gene sequences have a significant function, but there is still considerable risk in the retroviral vector method. Although it can be dangerous, this is most commonly used in patients with a small chance of survival, so the risk is not as ... Get more on HelpWriting.net ...
  • 37. Tuskegee Syphilis Study 1. What public health measures were widely used prior to the Sanitary Awakening? Before the sanitary awakening, the health measures used were mainly isolation techniques. Individuals were left in sectioned off areas to die or let their illness progress until the 1900s. In the 1900s during the Sanitary Awakening health officials start looking at disease by environmental conditions. 2. In what year did human population growths peak? In 1960 worldwide population growth reached an all time peak of 2.0. At this point demographers brought up the issue of carrying capacity. 3. The mean number of children born per woman in a particular population ranges between 1 and 10. This indicator is called what? Total fertility refers to the mean number ... Show more content on Helpwriting.net ... Local health departments are typically involved in providing preventative health services, communicable disease control programs, and community health education, among other activities. Which of the core functions of public health does this represent? The core function being represented is assurance. At this core function people are linked to personal health services needed, the medical workforce is checked to be competent, and the community is educated and informed of health issue that may arise. In addition, education and community health problems are identified and solved. 16. The Tuskegee Syphilis Study took place during which period of U.S. history? The Tuskegee Syphilis Study took place from 1932 to 1971 thus referring to the ears of the great depression and World War II. 17. What is a practice that best illustrates the principal of "Ethical Relativism?" Ethical relativism is the idea that deciding if an action is right or wrong depends on ones own societal normality. The practice of medicine demonstrates this principal. A doctor in one country may see giving a handshake as a welcome as right while in other countries this may not be considered the same. 18. World reaction to atrocities committed under Nazi Germany Led to what document? World reaction led to the development of the consent ... Get more on HelpWriting.net ...
  • 39. Rat Brain Case Study 1000 µg rat brain lysate was incubated with 50 µg construct overnight on a shaking incubator at 4°C. Samples were loaded on a polyacrylamide gel and processed for immunoblotting (see appendix) Immunoblotting for sacsin and JIP3 interaction Primary Sacsin and JIP3 antibodies (Santa Cruz cat. sc–515118 and sc–46663) raised in mice were added in a 1:3000 and 1:2000 ratio respectively to the corresponding membrane sections and incubated on a shaking incubator for 1 hour. Next, excess antibody was removed by washing 3 times with TBST. Secondary goat anti mouse HRP antibody (Cedarlane Laboratories cat. 115–035–146) was added in a 1:4000 ratio to 20 ml of 5% BSA in TBST. The membranes were washed 3 times with TBST. To image the proteins, 3 ml of ... Show more content on Helpwriting.net ... After three hours, the coverslips were carefully washed with 1X PBS at 37°C, and fixed for 20 minutes with 4% Paraformaldehyde in PBS. Immunofluorescence on HeLa cell lines The coverslips were washed with 1X PBS. Permeabilized for 5 minutes with 500 µl PBS + 0.2% Triton X–100, then washed again with 1X PBS. The cells were blocked for 1 hour with 2% BSA in PBS. Then the cells were incubated for 1 hour with primary LAMP1 antibody raised in mice at a concentration 1:50 (cat. ab25630 Abcam), diluted with 2% BSA in PBS. The coverslips were washed 3 times with 1X PBS and incubated for 45 minutes with secondary Alexa Flour 647 goat anti mouse antibody (cat. A21236 Invitrogen), at a concentration of 1:500, diluted in PBS + 2% BSA. The coverslips were then washed 3 times with 1X PBS. The coverslips were mounted using DAKO mounting media (cat. S3023), and imaging of the cells was performed using an LSM 710 (ZEISS) confocal fluorescence microscope. Results, add table for results but remove from text, no methods here at all, controls JIP3 interaction with sacsin domains To find the critical binding region of sacsin which interacts with JIP3, six well–characterized N– terminally GST–tagged sacsin deletion constructs, which include the three SIRPTs, DNAJ–HEPN, UBE3A binding site, and UBL domains (Fig.1), were expressed in a bacterial system and used in pulldown assays to test for JIP3 binding. These domains ... Get more on HelpWriting.net ...
  • 41. Dna Damage, Repair, And Dna Methylation Essay Introduction: Modification of damaged DNA seems to be an understudied subject, there is much to understand on the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by methylation but much of it is affected on a gene when silenced. When epigenetic modification has been implicated with cancer and aging it causes DNA methylation to also have an impact on the double strand of DNA analysis. Modification as such provoke deteriorating changes like aging found in multicellular organisms and DNA damage may magnify biochemical pathways that regulate a cells growth or control DNA replication with DNA repair. In the article "DNA Damage, Homology–Directed Repair, and DNA Methylation" written by Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may induce a DNA double–strand break within a genome as well as when DNA methylation is induced by homologous recombination that it may somewhat mark its reparation through a DNA segment and protect its cells against any unregulated gene expression that may be followed by DNA damage. The experiments used to demonstration how gene conversion can modify methylation pattern of repaired DNA and when that occurs methylation is able to silence the recombined gene. When exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an induced double strand break that can be found at a specific location or DNA sequence in where the ... Get more on HelpWriting.net ...
  • 43. Alzheimer's Disease Research Paper Alzheimer's disease Dementia is a broad category of brain diseases that cause long term loss of the ability to think and reason clearly that is severe enough to affect a person's daily functioning. Alzheimer's disease (AD) is the most common form of dementia. It primarily affects mainly older adults. Memory impairment is an essential feature of AD. Human central nervous system (CNS) is composed of neuron and glial cell ( astrocytes, oligodendrocytes, microglia) which act as structural support of brain and also contribute for formation of synapse. Microglia comprises 10% of the adult CNS. When there is tissue injury, trauma or pathogen, ramified microglia will undergo biochemical changes and turned into an activated microglia. The cell body ... Show more content on Helpwriting.net ... The experiment model Include a hypertrophic heart rat (HHR) and a normal heart rat (NHR). The HHR model showed no cardiac hypertrophy at maturity. Tissue from both HHR and NHR had been extracted. The cardiac weight index (CWI) was collected. The cardiomyocyte were then obtained via enzymatic procedure from both neonatal and adult NHR na HHR. Nuclei are then stained. Polymerase chain reaction (PCR) were used to search mRNA expression levels of ANP, BNP,MHC,p53, bax–1 and bcl–2 in ventricular tissues of HHR and NHR. Pl3K assay and western blot analysis were then performed. Lastly, a statistical analysis with value P<0.05 was obtained. The disadvantages of using this method were that the western blotting method is consider as a time consuming process that also require high technical demand. However, the interaction between antibodies and specific target protein can be clearly outlined. This will increase the sensitivity and specificity in the mixtures that contain mixture of ... Get more on HelpWriting.net ...
  • 45. Ultrasound Gene Delivery Ultrasound–based approached can facilitate drug and gene delivery by increasing the transport of therapeutic agents across the cell membrane and endothelial barrier. The function of ultrasound is that it can induce cavitation, which is a process of nucleation, growth, and oscillation for gaseous cavities. The cavitation can cause strong physical–chemical and biological effects in tissues1. Subsequently, the collapse of cavitation bubbles will disruptive the membrane of cells and tissues to enhance drug transport. Use the ultrasound to enhance cell permeability is a new methods for gene transfection. The increase uptake of DNA or RNA has been demonstrated by several investigators2– 1, 2–5. However, the further enhancement of gene transfection ... Show more content on Helpwriting.net ... So, the combination of ultrasound and transfection agents and Gene encapsulated in the agents could be a promising way to enhance gene delivery and transfection efficient significantly. The newly developed acoustically–active echogenic liposome in the form of a liposome associated with a gas bubble provides a platform for such applications. Liposomes composed of polyethyleneglycol, DSPC, and perfluoropropane gas provided a 60% increase in expression of green fluorescence protein plasmid DNA in rat eyes when ultrasound (1.2 W/cm2, 20 s, duty cycle 50%) was applied. The transfection efficiency of such bubble liposomes was higher than that of the popular lipofection agent, Lipofectamine 2000. Limitation: Ultrasound causes perturbation of cell membranes through the interaction of cavitation bubbles with the cells. This can lead to lysis of cells. Sonoporation appears to be injurious to the cells, thus, the condition of ultrasound should be careful controlled, to minimize side ... Get more on HelpWriting.net ...
  • 47. The Role Of Mir 125a And Mir 223 Experimental Design Aim1. To define the role of miR–125a and miR–223 in modulating Aicda and Prdm1 expression, CSR and plasma cell differentiation, by overexpression of miR–125a and miR–223 or inhibiting these microRNA, white mRNA "sponge" that competitively inhibit miR–125a or miR–223 using retrovirus construct. Aim1.1 Testing miR–125a overexpression can be performed via transforming a retrovirus vector construct into E.coli plasmid to amplify the vector, following by isolation of plasmids using specific Mini–prep Kit. Then, plasmids are transfected into Human Embryonic Kidney 293 cell line (HEK293 cell) or CH12F3 cell line. To determine the efficiency of transfection, we monitored green fluorescent protein (GFP) reporter construct expression by conducting either immunofluorescent assay or fluorescence–activated cell sorting (FACS) 14. We transduce the virus containing miR– 125a vector into primary B cells that is purified by mouse B cell isolation Kit and stimulated primarily with LPS followed by second stimulation using LPS and IL–4. Cells were collected 60 h later for qRT–PCR and RNA extraction, or 96 h later for surface Ig analysis by FACS. The stimulated B cells are stained with CD19 and CD138 to measure the percentages of switched B cells by FACS. We predict a reduction in IgG1, IgG3, and CD138 populations due to the impede function in CSR and plasma cell differentiation due to miR–125a expression, which target Aicda and Prdm1. In addition, RNA extraction will be ... Get more on HelpWriting.net ...
  • 49. The Rna Interference ( Rnai ) Pathway The RNA interference (RNAi) pathway is an important biological system that is utilised by many organisms as a method of regulating gene expression. But what exactly is RNAi? And what is the importance of its use and application in biology? RNAi is a cellular process that actively silences specific genes. This method of post–translational gene regulation has been observed in many organisms including plants, fungi, some bacteria and animals. RNAi inhibits protein synthesis of the target transcript by the use of small RNA molecules that target messenger RNA (mRNA) within the cytoplasm following transcription by an RNA polymerase. RNAi occurs during translation of the mRNA by inhibiting the action of Ribosomes that catalyse this process within ... Show more content on Helpwriting.net ... Upon binding the two strands of siRNA are separated, with one strand – known as the guide strand – remaining bound to RISC and the other strand being degraded. The guide strand then directs RISC towards its target mRNA for cleavage (Zamore, et al. 2000). This mechanism is extremely precise as it relies on complimentary base pairing between the bound siRNA and its target mRNA. This complementarity allows RISC to bind to the target mRNA where the Argonaute protein catalyses it's cleavage, resulting in degradation of the target mRNA (Fig. 1). This mechanism inhibits translation of the protein for which the target mRNA codes for and therefore silences that specific gene. RNAi can also involve the use of coded short hairpin RNAs (shRNAs), which are larger dsRNA molecules containing a loop section which folds back on itself to form a double stranded molecule contains a sense and antisense strain. The shRNAs are cleaved into siRNAs when they bind to RISC and can also found in examples of exogenous introduction such as viral or experimental insertion. The ability to post translational control of gene expression with a precise sequence specific manner has been exploited experimentally in researching gene knockdown and loss of function gene analysis. In 2006, Andrew Fire and Craig Mello were awarded the Nobel Prize in Physiology or Medicine for their work in RNAi in Caenorhabditis elegens. Their 1998 paper demonstrated RNAi of endogenous ... Get more on HelpWriting.net ...
  • 51. Dysbindin-1 Case Study Dysbindin–1 promotes Drp1 oligomerization Mitochondrial fission requires dynamin–related protein 1 (Drp1). Drp1 is a large GTPase that can assemble into oligomers with enhanced GTPase activity. Drp1 is predominantly located in the cytosol and can be recruited to mitochondria to mediate mitochondrial fission by forming an oligomeric ring around the mitochondrial tubule to constrict and sever mitochondrial outer membranes 38–43. Having found that dysbindin–1 regulates mitochondrial fission, we proceeded to test whether dysbindin–1 is involved in the association of Drp1 with mitochondria. Cultured hippocampal neurons (DIV14) were transfected with the mitoDsRed plasmid along with the dysbindin–1 siRNAs, scrambled siRNAs, dysbindin–1C or –1A ... Show more content on Helpwriting.net ... The mitochondrial fraction of sdy hippocampi contained more monomeric Drp1 and fewer higher– order Drp1 oligomers than that of WT hippocampi, while the whole cell lysate of WT and sdy hippocampi had comparable amounts of monomeric and oligomeric Drp1 (Figure 4A–C). These results indicate that dysbindin–1 is required for Drp1 oligomerization on mitochondria. Given that dysbindin–1C promotes the recruitment of Drp1 to mitochondria (Figure 3A–C), we assessed Drp1 oligomers in cultured hippocampal neurons transduced with lentivirus expressing dysbindin–1C or –1A. Overexpression of dysbindin–1C, but not –1A, increased higher–order Drp1 oligomers and decreased monomeric Drp1 in the mitochondrial fraction (Figure 4D–F). Hence, dysbindin–1C promotes Drp1 oligomerization on mitochondria. How does dysbindin–1 regulate Drp1 oligomerization? Dysbindin–1 has been detected on mitochondrial outer membranes by electron microscopy 28. It is possible that dysbindin–1 binds to Drp1 to enhance its oligomerization. To test this possibility, we transfected HEK–293 cells with YFP–Drp1 along with HA–tagged dysbindin–1C or –1A, and used an anti–HA antibody to immunoprecipitate dysbindin–1 at 2 days after transfection. Drp1 was detected in the immunoprecipitation product from cells transfected with dysbindin–1C, but not with –1A plasmid (Figure 4G), indicating that ... Get more on HelpWriting.net ...
  • 53. Glutamate Transmission Genetically encoded fluorescent probe for visualizing glutamate neurotransmission in organotypic hippocampal slices Neurotransmission plays an essential role in communication between neurons. In such process, neurotransmitters are released by the axon terminal and those bind to a receptor on the dendrite of another neuron. Among all neurotransmitters, glutamate is the most significant one in vertebrates. It is used in a large percentage of circuits in the cerebral cortex that support learning and complex behaviors. Modifications to transmission of glutamate will affect the function of neural circuits on a large scale in the brain.[1] Glutamate transmission can be studied at the level of individual dendritic sites. In this case, recent studies ... Show more content on Helpwriting.net ... I will image these cells and using two–photon glutamate uncaging, individual spine synapses will be stimulated. This stimulation involves releasing glutamate close to the spine, in which I would expect to see an increase in fluorescence when glutamate binds to the genetically encoded sensor. Being able to analyze the change of fluorescence is important to this experiment because the resulting measurements describe the kinetics of the tested constructs. I have been working on this project since the summer and we obtained data of these candidate sensors in HEK cells. From this data, I selected the ones that showed robust signals and tested them in ... Get more on HelpWriting.net ...
  • 55. Circumventing Traditional Bottlenecks In Glioblastoma... Circumventing traditional bottlenecks in glioblastoma genetic therapy: ultra–pH sensitive nanodelivery vehicles RNAi–based modalities offer sequence–specific knockdown of tumor metabolism, presenting a novel alternative to ineffective treatments. This study investigated a pH sensitive siRNA nanoparticle which circumvents traditional barriers in genetic therapy by promoting intracellular delivery, reducing cytotoxic off–target transfections, and facilitating endosomal escape of siRNA. Acyl–CoA synthetase 5 (ACSL5) was explored as a target for knockdown of glioma viability. Poly(2–(diisopropylamino) ethyl methacrylate) (PDPA) nanoparticles were synthesized via nanoprecipitation, measured for cell uptake with fluorescence microscopy, ... Show more content on Helpwriting.net ... PDPA NP–siACSL5 inhibits glioblastoma proliferation via fatty acid oxidation disruption siACSL5–PDPA NP transfected U87MG cells and were measured for growth and survival inhibition via Ki67 staining and fluorescence microscopy. Cells transfected with naked siRNA exhibited 55% glioma proliferation as compared to the control group which amassed a 78% proliferation rate (p>0.05), exhibiting the poor efficacy of non–encapsulated siRNA. In contrast, PDPA NP–mediated inhibition of cell proliferation resulted in less than 22% proliferation and five fold inhibition of tumor cell proliferation compared to the control group (Fig. 8; p<0.05). siACSL5–PDPA nanoparticle complexes disrupt the ACSL5–Midkine pathway, leading to activation of downstream pro–apoptotic proteins such as caspase–3 and simultaneous knockdown of proteins associated with cell cycle survival such as Midkine, mTOR, and PKB. The two–pronged knockdown of both glioma proliferation and survivability via forked pathways offers an effective therapeutic solution to combat tumor viability. Conclusions The PDPA NP formulation serves as an efficient transfection platform due to its ability to bind to siRNA and condense/stabilize large ... Get more on HelpWriting.net ...
  • 57. Transfection Lab Report Transfection of pCas9–GFP with HEK293 cells Introduction Transfection is the insertion of plasmid DNA into eukaryotic cells. Transfection is used to look at the expression of a gene inserted in a cell. For this laboratory, the eukaryotic cells used were HEK293 cells. 293 cells are derived from human embryonic kidney cells. The reagent used to transfect pCas9–GFP into 293 cells was polyethylenimine (PEI). PEI is a cationic polymer and is the transfecting agent used to introduce pCas9–GFP into the 293 cells via endocytosis and thus releasing the DNA into the nucleus. OptiMEM was also used for the transfection of the cells; it provided a biological environment to the eukaryotic cells, thus increasing the rate of transfection. Following the ... Show more content on Helpwriting.net ... Next, in figure 2, the 96–well plate shows a change of color after the incubation of BCA reagent and both dilutions. Wells 9 A–B contained the 1:5 dilution, while wells 9 E–G contained the 1:10 dilution. The BSA standards are found in wells 1, 3, and 5 A–G. Wells 1, 3, and 5 H were used as blanks; they only contained 40 μl of IP lysis. The results obtained from the spectrophotometer were used to create the graph and obtain the equation found in figure 3. The equation was used to find the concentration of the protein, which is 3.45 μg/μl. Figure 4c shows two bands obtained from the Western blotting performed on GFP; the bands were compared to the protein scale (Figure 4a). The first band is at ∼25kDa and the second band is at ... Get more on HelpWriting.net ...
  • 59. Mammalian Cell Culture Lab Answers Mammalian Cell Culture Lab Manual Student guide of Cell Biology Laboratory Techniques 2012 Jay M. Feldman Cell Biology Professor Lagier 11/26/2012 Mammalian Cell Culture Lab Manual Student guide of Cell Biology Laboratory Techniques 2012 Jay M. Feldman Cell Biology Professor Lagier 11/26/2012 Table of Contents I. Introduction * About the author page 2 * Preface page 3 II. Laboratory Techniques * Cell Culture Technique page 4–5 * Sterile Technique page 6–7 * Media Preparation page 8–9 * Seed cells page 10 * Subculture page 11–12 * Cell count page 13–14 * Cryopreservation page 15 III. Laboratory Experiments * Intentional Contamination page 16–17 ... Show more content on Helpwriting.net ... A. They are immortal B. They are epithelial cells C. They are cancer cells D. All of the above 3) What is an advantage of cell culture? A. Control the physiochemical environment of an experiment B. It is more fun than anti cell culture C. It is easier than anti cell culture D. All of the above Answers: 1) C 2) D 3) A Introduction to Sterile Technique * What is Sterile Technique?
  • 60. Sterile technique is the process of executing cell culture procedures without introducing contaminating microorganisms (bacteria) from the environment * Sterilize vs Sanitize The difference between sanitization and sterilization is that when you sanitize something, you are getting rid of MOST of the live bacteria. It takes about 30 seconds and we perform this using 70% ethanol or Lysol. When we sterilize something, we make free of ALL live bacteria. Sterilization is done with an autoclave or by gas sterilize (this is not done in lab). –Something that is sterilized is also considered sanitized, but if something is sanitized, it is not sterile. * Personal Hygiene By definition, personal hygiene is the act of grooming, cleaning, and looking after our bodies. In cell biology lab, personal hygiene will be necessary in order to keep contaminants from your body from reaching your cells. By having good hygiene, you will be able to reduce most of the risk of spreading bacteria by getting rid of it before you ... Get more on HelpWriting.net ...
  • 62. Biology Case Study Materials and methods The levels of microRNA–22–3p (miR–22–3p) and TP63 were analyzed by RT–qPCR and/or Western blot. Cell proliferation was measured using MTT assay. Cell invasion and migration were evaluated using Transwell assay chambers. Prediction of a regulatory relationship between microRNA–22–3p and 3'–UTR of TP63 mRNA was performed by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. OC cell lines Lung cancer cell lines H292, PC–9, CL1–5, A549 and normal NHBE cells were used in this study. All of Lung cancer and normaL ceLL Lines – were provided by ATCC. H292, PC–9, CL1–5, A549 and NHBE cells were cultured in RPMI–1640 medium supplemented with 10% fetal bovine serum (FBS). MiRNA and RNA interference ... Show more content on Helpwriting.net ... The experiments were repeated thrice. Cell migration and invasion assays In vitro cell migration and invasion were examined using the Boyden Chamber Assays. For the cell migration assay, 30000 cells in 100 μl DMEM medium devoid of fetal bovine serum (FBS) and a given concentration of cetuximab were cultured on a polycarbonate membrane insert coated with fibronectin in a Boyden Chamber (Essen BioScience, UK). In the lower chamber, 500 μl DMEM with 10% FBS was added as chemoattractant. After the cells were incubated for 6 h at 37 °C in a 5% CO2 atmosphere, the insert was rinsed using PBS and cells on the upper side of the insert were gently removed using a cotton swab. Subsequently, cells that adhered to the lower side were fixed with methanol, stained with crystal violet solution and counted under a microscope in five predetermined fields (×200). All assays were independently repeated at least thrice. The same protocol as described above was used for the cell invasion assay, except that the transwell filter membranes were pre–coated with 24 μg/μl matrigel (R&D Systems, Inc., Minneapolis, MN, USA) and the cells were incubated for 8 h at 37 °C in a 5% CO2 atmosphere. RNA isolation, reverse transcription and RT–qPCR RNA was extracted from the OC cells using the Trizol reagent. For miR–22–3p, RNA was transcribed into complementary DNA and amplified using miRNA PrimeScript RT Enzyme Mix kit according to the manufacturer's instructions ... Get more on HelpWriting.net ...
  • 64. Essay On Cosisp I believe that CRISPR is going to, and already is revolutionizing medicine. The way we fight disease, cure cancer, and maybe even create new humans. The editing techniques I'm about to mention allow scientist to modify DNA sequences in the human body and. So what is CRISPR? We first have to understand where CRISPR comes into play, and that is genomic editing. Genome editing is where engineered nucleases are either inserted, replaced, or deleted in a living organism's genome, and genomic editing is done with the help of the system CRISPR. The CRISPR cas system is an RNA guided nuclease system for targeted introduction of doubled stranded DNA cleavage. It was originally discovered in bacteria as an acquired defense against foreign ... Show more content on Helpwriting.net ... Other vector variations are also available to provide added convenience, such as GFP for transfection monitoring or lenti–backbone for viral delivery. For researchers who perform mRNA microinjection or mRNA transfection. The applications for CRISPR Cas 9 are endless. There are two types of editing that are commonly used: homologous recombination and non– homologous end joining(NHEJ). Homologous recombination utilizes a repair template The desired changes, such as gene knock out, gene tagging, and specific mutations, are flanked by left and right homologous arm sequences. Upon double cross over the desired changes integrate into the genome. In non–homologous end joining the ends are joined together and introduce random mutations. Both types are used to remedy double strand breaks (DSBs), but homologous recombination is highly preferred over NHEJ because this type of editing is extremely error prone. It can delete or add genetic information and cause chromosomal translocations. So why aren't we using these mechanisms on humans in order to control our genetic makeup? In the medical field we see great opportunity to treating and even curing some of the most horrible of disease, yet we are still hesitant. Just as easy, as it will soon be, to edit genes for healing purposes scientists, researchers, and physicians will have to embrace the uncertainty that comes with it. The uncertainty of incorrectly placing DNA, or deleting/adding DNA that ... Get more on HelpWriting.net ...
  • 66. Western Blotting Second, for the western blotting, the primary antibodies that used to recognize the protein of interest (FLAG–PTEN) are rabbit anti–PTEN antibodies and the secondary antibodies that bind to the primary antibodies and convalently linked substrate to produce visible signal are goat anti–rabbit conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used to immobilize protein of interest are anti–FLAG monoclonal antibodies from morine cell. As introduction above, those antibodies come from different species. In order to be successful in western blotting, the primary antibodies recognize the protein of interest as well as bind to it. Consequently, rabbit anti–PTEN antibodies were used to bind to FLAG–PTEN. Meanwhile, goat anti–rabbit conjugated HRP antibodies, the secondary antibodies, ... Show more content on Helpwriting.net ... As a result, the calculated FLAG–PTEN molecular weight is nearly 72KDa. When comparing the previous research, the theoretical molecular weight of PTEN is 47KDa, the experimental ones is 25KDa more. The reasons would be [1] since the PTEN was tagged with the FLAG tag and the FLAG tag itself has its own weight. Although FLAG tag is small in size and contains eight amino acid, it would affect the finial molecular weigh of FLAG–PTEN; [2] The FLAG–PTEN were expressed in the mammalian cell system, its original environment. Therefore there would be some proteins that interact with PTEN naturally in mammalian cell, referring as interacting partner, and they bind to FLAG–PTEN in the cell lyaste sample then precipitate together in the IP. Those interacting proteins would increase the molecular weight of FLAG–PTEN by forming a complex and result in a higher weight band; [3] The resolving ability of the SDS–PAGE is not enough for the completely separation of the sample, as some of the lower weight markers were compacted in the ... Get more on HelpWriting.net ...
  • 68. The CRISPR/Cas9 System Abstract The CRISPR/Cas9 system is an accurate, cheap, and fast way to edit genes. It is used to edit parts of the genome by removing, adding, or altering some sections of the DNA sequence. The CRISPR/Cas9 system was used with HIV cell lines and RNA–directed gene editing to prevent new HIV–1 infections. Both experiments resulted in the stopping of the reactivation of the HIV infection. HIV (Human Immunodeficiency Virus) puts its own genome directly into a host cell during the time of infection (Ebina et al, 2013). This can lead to AIDS (acquired immunodeficiency syndrome) if it is left untreated. AIDS is caused by a retrovirus. HIV can remain dormant for years once its DNA is in the host cell (Fessenden, 2015). HIV infects ... Show more content on Helpwriting.net ... By using the CRISPR/Cas9, it is possible to alter DNA to correct genetic diseases in animals and modify DNA sequences in embryonic stem cells (Rogers, 2016). This opened up the process of genome modifications in humans through the germ line (sperm and egg) (Rogers, 2016). Doudna became interested in the CRISPR and found out it is part of the bacterial immune system. It all started with RNA sequences from invading viruses that become part of the bacterial genomes. From there the viral sequences stay as DNA in the spacers between the short repeating blocks of bacterial DNA sequences (Rogers, 2016). When the virus attacks the bacterial cell again, the spacer DNA is converted to RNA. A Cas9 enzyme and a second RNA molecule attach themselves to the new RNA and then searches for a matching strand of viral DNA (Rogers, 2016). The Cas9 cuts the viral DNA which prevents the virus's replication. Charpentier and Doudna found that the guide RNA sequence could change the direction of the Cas9 to a precise DNA sequence. By this finding, it transformed the genome engineering and could be treatments for human diseases (Rogers, ... Get more on HelpWriting.net ...
  • 70. Bb Ap Essay APPROACH 3 Towards a diagnosis of Bb–Ap co–infection and Lyme neuroborreliosis Currently, the effective diagnosis of Lyme disease is challenging. Usually, a two–steps serological test approach is recommended, using ELISA and Western Blot, but a high incidence of false– negative results is observed. Molecular diagnosis based on PCR are not recommended since Bb DNA may persist in the host long after the bacterial elimination. Consequently, there is an urgent need to develop a specific diagnostic tool for Lyme disease, including during Bb–Ap co–infection and Lyme neuroborreliosis. 3.a. Identifying targets of Bb–Ap co–infection In order to identify possible diagnostic targets, I will take advantage of the in vivo Microbial Antigen Discovery (InMAD) approach23. This methodology, especially useful vis–à–vis of pathogens that are present in very small quantities in the blood, is based on the immunization of mice using filtered serum from infected animals, in order to promote an immune response against circulating microbial antigens, but not against cell–related antigens. Then, the immunized sera are tested against bacterial lysates in 2D–western blots and positive spots are identified by mass spectrometry as potential diagnostic candidates. 3.b. Developing antibodies against Bb–Ap ... Show more content on Helpwriting.net ... In the unlikely scenario that this double–transfection might alter viability/virulence of these microorganisms, I will consider the single–transfection with only bioluminescent genes. The double–transfection is more challenging and risky, however, it generates completely versatile microorganisms to be used in a wide range of image–based techniques, consequently, fully justifying this high–risk/high–reward strategy. Importantly, I used a similar strategy to produce bioluminescent/fluorescent Trypanosomatids parasites24, underscoring the presumable feasibility of this experimental ... Get more on HelpWriting.net ...
  • 72. Transfection Lab Abstract: This lab was conducted to determine the efficiency of transfection in HEK293 cells using cell culture and lipofectamine by growing the HEK293 cells out in growth medium, and then passaging them into 24 well plate at 50% confluency. After the passaging of the cells, and aspirating the media after letting the cells grow in the wells for 24 hours, then a master–mix of lipofectamine and GFP plasmid DNA was added to 500 microliters of stock DMEM, and then adding 100 microliters of the master–mix to each well, and allowing then to grow overnight.Then the next day accutase was added to dislodge the cells and placed on a side to view them under a UV microscope to find the efficiency of the transfection. The experimental had the same procedure ... Show more content on Helpwriting.net ... Our hypothesis was that there would be efficiency would be lower, and it was supported by the results obtained in the lab run. The results that we received are in correlation to a paper by Thermo Fisher, it makes sense because Thermo were the ones we received the procedure and the materials from to complete this lab. This is because the procedure is optimized for the highest efficiency. The dependent variable is the efficiency of the transfection of the HEK 293 cells, and the independent variable is the amount of Lipofectamine 2000 used in the master–mix. The lab gave the result that the efficiency of the control was 39.6% and the experimental 30.0%, showing a difference in the efficiencies of the two groups. It was found in a t–test that the results were statistically significant, the p value is lower than 0.05, and the p–value was 0.0000333530994, which is less than .05. There were errors in goring out of cells ranging from contamination and the cells not attaching and growing, there was an error in the first time doing transfection when the instructions were not clear of how much GFP plasmid was needed in the mastermix, too much was used and there was not enough plasmid left to fill and the wells with medium. Future implications of this research would be to grow out more complex cells in culture and ... Get more on HelpWriting.net ...
  • 74. Specific Aim 2: To Determine If Gleevec Is Effective In Specific Aim 2: To determine if Gleevec is effective in inhibiting in vivo cell proliferation in TNBC cells with overexpressed Arg A recombinant plasmid containing a viral promoter driving Arg expression will be inserted into TNBC cells. Another empty recombinant plasmid will be inserted into different TNBC cells. Another plasmid containing a promoter driving Arg expression will be inserted into another group of TNBC cells. Soon after, an immunoblot will be run using these three different TNBC groups' cells to ensure the proper transfer of plasmid. The proteins we will test for include Arg, Abl, actin, and Ras–MAPK. Actin is again serving as a positive control, while the expression of Ras–MAPK proteins can act as a quantitative ... Show more content on Helpwriting.net ... Abl is known to mutate its kinase domain so that Gleevec cannot effectively inhibit it and cellular proliferation (Lovera et al., 2015). A western blot will be run to determine the extent this mutation will affect our experiment at the beginning and 4 months into our experiment. Some tumors from all three cell lines will have their cells lysed and run through the gel, which will test for both Abl and Abl–Gleevec compound, as well as a major substrate for Arg–Abl, ArgBP2. Results– The western blot containing the three different cell lines expressed an equal amount of actin expressed in all three lanes. This served as a control to ensure the plasmids were inserted correctly and there was no interference of any other gene. The lane with cells from mice group 1 showed darker bands on lines corresponding to the proteins Arg as opposed to the lane with mice group 2 cells. The bands corresponding to the protein Abl appeared to be the same on both lanes, demonstrating that Abl was not being accidentally overexpressed as well. These pieces of information elucidated to us that the Arg gene was being successfully overexpressed in line 1. The bands corresponding to Ras–MAPK were darker for mice group 1 than for group 2, demonstrating that this signal transduction pathway for cell proliferation has been downregulated with the overexpression Arg. ... Get more on HelpWriting.net ...
  • 76. T Cell Gene Therapy : The Center Of Research With Stem... The CCR5 co–receptor is not only the center of research with stem cell transplant, but has also been a recent target of gene therapy research. Gene therapy is a fairly new technology where genes (edited or normal) are transplanted in humans to produce a specific response. One avenue of research that is currently being studied is T cell gene editing focused on the CCR5 delta32 mutation in HIV infected patients using the CRISPR/cas9 system. There is some research using TALENS, but evidence with CRISPR/cas9 will be discussed here (Ye, 2014). The CRISPR technique that edits gene sequences has been also researched for use in Hepatitis B, Epstein Bar Virus, Malaria, and Human Papilloma Virus. Simply put, the CRISPR/cas9 system can be ... Show more content on Helpwriting.net ... The researchers created and studied the best way for the CRISPR/cas9 system to be delivered into CD4 cells. The best delivery mode for the CRISPR/cas9 was assessed to be an adenovirus known as the Ad5F35, and the optimal time for exposure for maximal gene editing was found to be 8 days. The CRISPR/cas9 was introduced to the cells by the Ad5F35 adenovirus and eight days passed to allow the system to modify the genetic makeup. Following transduction of the CD4 cells with the genetically engineered CCR5 mutation, the cells remained HIV resistant. This study claims to be the first to successfully use an adenovirus to transduce genetically modified DNA into CD4 cells to provide resistance to HIV. In theory, these modified CD4 cells can then be transplanted in HIV patients so that their fighter cells will be resistant to HIV infection. (Li, 2015). Another recent study involved transplanting these modified T cells into mice using the CRISPR/cas9 to see if the engineered cells would transfer resistance to HIV. After transplanting the T cells and exposing the mice to HIV, the mice tested resistant to the virus (Zu, 2017). The author in the initial CRISPR study, Li, believed it to be important to create a way to genetically change the CXCR4 co–receptor as well as the CCR5 co–receptor so that the virus cannot utilize any receptor to enter the host cell (Li, 2015). In a study done in 2017, scientists developed a way to alter the genetic makeup of the CXCR4 co–receptor. Instead of ... Get more on HelpWriting.net ...
  • 78. Transgenic Mice Essay Transgenic Mice, Care and Handling Double transgenic mice express a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1–dE9) were purchased from The Jackson Laboratory (004462, Bar Harbor, ME, USA). All animal treatments were approved by the Animal Ethics Committee of Tsinghua University. Mice had access to pre– filtered sterile water and standard mouse chow and were housed under a reversed day–night rhythm in accordance to local legislation on animal welfare. Cell Lines and Reagents A172 and U251 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and the Chinese Academy of Sciences (Kunming, Yunan, China), respectively. Cell cultures were maintained in ... Show more content on Helpwriting.net ... Transfection and selection was pursuant to previous description [40]. The empty vector served as the negative control. For the EGFP–LC3 cell line, the pEGFP–LC3 overexpression plasmid was previously prepared in our lab and used for cell transfection, followed by G418 (100 μg/ml) selection. Transient Transfection Cells were transfected with the miR34a, an antisense miR34a inhibitor (anti–miR34a), siSH3GL3 (5′–GUAAUGCAUUGAUAGAAGU–3′), siCTSB (5′– AGAGAGUUAUGUUUACCGA–3′), siSH3GL2 (5′–GAACCAAGCUAGAUGAUGA–3′), siSH3GL1 (5′–GCAAGGCGGUGACAGAAGU–3′), and siPPP1R18 (5′– GCUCGAGAAUGGACACCCA–3′), using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) according to the manufacture's recommendation. A scramble siRNA (5′– UUCUCCGAACGUGUCACGUTT–3′) was used as negative control. 48 or 72 h after transfection, cells were treated as indicated. Western Blot (WB) Cells were lysed with Laemmli buffer, and each lysate sample was loaded into a polyacrylamide gel and separated by electrophoresis at 30 mA following by transfer onto PVDF membranes (Millipore). Membranes were blocked for 1 h at room temperature using 5 % skim milk in Tris–Buffered Saline with 0.1 % Tween–20 (TBST). WB analysis was ... Get more on HelpWriting.net ...