This document describes research into producing recombinant adeno-associated virus (rAAV) using serum-free suspension cultures of HEK 293 cells. The researchers investigated the effects of different DNA, polyethyleneimine (PEI) transfection reagent, and cell concentrations on rAAV yields. They found that transfecting at a cell concentration of 3x10^6 cells/mL resulted in a 2.5-3.5 fold increase in infectious virus particles (IVP) and genomic particles (Vg) compared to 1x10^6 cells/mL. When testing different PEI:DNA ratios, IVP ranged from 1.19x10^8 to 1.03x10^9 IVP/
This document describes the construction of an expression vector called pET-DB that contains a downstream box (DB) sequence to enhance protein expression in E. coli. The DB sequence matches the anti-DB sequence in 16S rRNA and facilitates translation initiation. Four genes were cloned into pET-DB and a control vector without the DB to test expression levels. Results showed that pET-DB increased protein expression by 35-70% compared to the control vector. The improved expression and ability to easily purify proteins with a His-tag make pET-DB a useful vector for high-level protein production in E. coli.
A new system called the "admid system" has been developed for efficiently generating recombinant adenoviruses in E. coli. The system uses Tn7-mediated transposition to insert expression cassettes into an adenoviral genome plasmid (admid) maintained in E. coli. Transfer vectors containing the expression cassette flanked by Tn7 elements transpose the cassette into the E1 region of the admid. Recombinant admids produce infectious adenovirus after transfection into producer cells. The system generates pure, clonal adenovirus stocks without multiple rounds of purification. It allows rapid, high-throughput production of recombinant adenoviruses for gene therapy and other applications.
This document contains a sample exam for the GATE biotechnology exam from 2000. It includes 31 multiple choice or short answer questions testing knowledge of topics like restriction enzymes, DNA structure, gene expression, cloning, PCR, and more. The questions range in difficulty from recalling basic concepts to applying knowledge through calculations.
This document describes a protocol for one-step targeted gene deletion in Candida albicans haploids. C. albicans is an important human fungal pathogen that was previously thought to exist only as a diploid. However, the discovery of viable haploid strains of C. albicans enables more efficient gene deletion through a single transformation step rather than the previous two-step process required in diploids. The protocol uses URA3 flipper cassettes containing flanking regions of homology to the target gene for deletion. Transformants are screened by colony PCR to identify those with correct gene deletion and assess ploidy. The protocol enables more rapid functional analysis of C. albicans genes.
This document reports on a laboratory project investigating the role of the non-coding RNA rli60 in Listeria monocytogenes. The student constructed an rli60 knockout strain of L. monocytogenes and examined gene expression and metabolism compared to the wild type strain when grown in different media. Quantitative PCR results showed that rli60 is involved in down-regulating the ilv operon, which encodes for branched amino acid biosynthesis. Growth curve and luminescence assays indicated normal bacterial growth and hly virulence gene expression in the rli60 mutant strain. In summary, this project found that the non-coding RNA rli60 regulates metabolic gene expression but does not affect growth or a key virulence factor in L. monocytogenes
Improved vector design eases cell line development workflow in the CHOZN GS-/...Merck Life Sciences
This poster was presented at ESACT meeting in 2017 in Lausanne, Switzerland. Cell line development for production of monoclonal antibody therapeutics requires an expression vector encoding both the heavy and light chains of the antibody. When expression of the heavy and lights chains is driven by the same promoter, the sequence redundancy can be problematic for verifying the vector sequence, copy number and insertion site in the host cell genome. This poster describes the work done to identify an expression vector that maintains a high level of antibody expression but lacks the sequence similarities, easing the cell line development workflow.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This document describes the construction of an expression vector called pET-DB that contains a downstream box (DB) sequence to enhance protein expression in E. coli. The DB sequence matches the anti-DB sequence in 16S rRNA and facilitates translation initiation. Four genes were cloned into pET-DB and a control vector without the DB to test expression levels. Results showed that pET-DB increased protein expression by 35-70% compared to the control vector. The improved expression and ability to easily purify proteins with a His-tag make pET-DB a useful vector for high-level protein production in E. coli.
A new system called the "admid system" has been developed for efficiently generating recombinant adenoviruses in E. coli. The system uses Tn7-mediated transposition to insert expression cassettes into an adenoviral genome plasmid (admid) maintained in E. coli. Transfer vectors containing the expression cassette flanked by Tn7 elements transpose the cassette into the E1 region of the admid. Recombinant admids produce infectious adenovirus after transfection into producer cells. The system generates pure, clonal adenovirus stocks without multiple rounds of purification. It allows rapid, high-throughput production of recombinant adenoviruses for gene therapy and other applications.
This document contains a sample exam for the GATE biotechnology exam from 2000. It includes 31 multiple choice or short answer questions testing knowledge of topics like restriction enzymes, DNA structure, gene expression, cloning, PCR, and more. The questions range in difficulty from recalling basic concepts to applying knowledge through calculations.
This document describes a protocol for one-step targeted gene deletion in Candida albicans haploids. C. albicans is an important human fungal pathogen that was previously thought to exist only as a diploid. However, the discovery of viable haploid strains of C. albicans enables more efficient gene deletion through a single transformation step rather than the previous two-step process required in diploids. The protocol uses URA3 flipper cassettes containing flanking regions of homology to the target gene for deletion. Transformants are screened by colony PCR to identify those with correct gene deletion and assess ploidy. The protocol enables more rapid functional analysis of C. albicans genes.
This document reports on a laboratory project investigating the role of the non-coding RNA rli60 in Listeria monocytogenes. The student constructed an rli60 knockout strain of L. monocytogenes and examined gene expression and metabolism compared to the wild type strain when grown in different media. Quantitative PCR results showed that rli60 is involved in down-regulating the ilv operon, which encodes for branched amino acid biosynthesis. Growth curve and luminescence assays indicated normal bacterial growth and hly virulence gene expression in the rli60 mutant strain. In summary, this project found that the non-coding RNA rli60 regulates metabolic gene expression but does not affect growth or a key virulence factor in L. monocytogenes
Improved vector design eases cell line development workflow in the CHOZN GS-/...Merck Life Sciences
This poster was presented at ESACT meeting in 2017 in Lausanne, Switzerland. Cell line development for production of monoclonal antibody therapeutics requires an expression vector encoding both the heavy and light chains of the antibody. When expression of the heavy and lights chains is driven by the same promoter, the sequence redundancy can be problematic for verifying the vector sequence, copy number and insertion site in the host cell genome. This poster describes the work done to identify an expression vector that maintains a high level of antibody expression but lacks the sequence similarities, easing the cell line development workflow.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
Universal and rapid salt extraction of high quality genomic dna for pcr-based...CAS0609
This document describes a simple and universal method for extracting high-quality genomic DNA from a variety of organisms including plants, fungi, insects, and shrimp. The method uses a salt-based homogenizing buffer and SDS to extract DNA from as little as 50mg of fresh tissue. The extracted DNA is of sufficient quality and quantity to be used in PCR, restriction digestion, and other molecular techniques. The method is fast, inexpensive, and does not require expensive equipment, making it suitable for laboratories with limited resources. Test results demonstrated the method successfully extracted high molecular weight DNA from many diverse organisms without modification, indicating its universal applicability.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
The document summarizes research on the Monascus genome project and related genetic studies. Key points include:
1) The Monascus genome was sequenced to 8x coverage, revealing various genomic features. A Monacolin K biosynthesis gene cluster was identified containing genes for polyketide synthases and other enzymes.
2) An efficient method for genetic transformation of Monascus pilosus was developed using aurintricarboxylic acid.
3) Disruption of the mokA gene and overexpression of the mokH transcription factor gene resulted in loss of and increased Monacolin K production, respectively.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document summarizes work done on culturing crocodile cell lines and cloning the parc gene from Pseudomonas keratitis. Primary crocodile cell lines were established from various organs and immortalized using hTERT. The parc gene was cloned from mutant and wild-type Pseudomonas strains and will be expressed and crystallized to study its role in quinolone antibiotic resistance.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
Recombinant DNA technology involves isolating and amplifying specific DNA sequences and incorporating them into vector molecules like plasmids or bacterial viruses. This recombinant DNA is then propagated in organisms like E. coli. Genetically engineered bacteria can produce important human proteins like insulin, growth hormone, and clotting factors. Restriction enzymes and DNA ligase are used to cut and join DNA fragments to create recombinant molecules.
This study demonstrates for the first time the production of recombinant human growth hormone (rhGH) from Bacillus subtilis. A hybrid gene containing the signal sequence of the B. licheniformis serine alkaline protease gene and the cDNA encoding hGH was cloned into the pMK4 plasmid and expressed under the deg promoter in B. subtilis. The rhGH produced was secreted and confirmed to have the correct mature hGH sequence. The highest rhGH concentration of 70 mg/L was obtained at 32 hours with a yield of 9 g/kg. Fermentation characteristics differed between B. subtilis containing the hybrid gene versus the plasmid alone. This expression system could be applicable for heterologous protein production from Bac
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
The document summarizes a study on gene expression during regeneration of Drosophila wing imaginal discs. Microarray analysis found over 1,000 genes differentially expressed between cut and intact discs in the first 24 hours after wounding, indicating a strong initial response to injury. Many genes involved in apoptosis, stress response, cytoskeletal activity and JNK signaling showed increased expression. Between 24-72 hours, expression returned toward normal levels as discs resumed activities interrupted by wounding. The study identifies signaling pathways and gene categories important for disc regeneration.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
This thesis examines the role of the transcription factor Gli3 in regulating neural stem cell proliferation and neuronal fate specification in the subventricular zone (SVZ). The author finds that:
1) Expression of the Gli3 repressor form is higher in dorsal SVZ neural stem cells, while expression of the activator form Gli1 is higher in ventral SVZ stem cells.
2) Conditional ablation of Gli3 in dorsal SVZ stem cells results in their progeny adopting aberrant positions deeper in the olfactory bulb compared to controls.
3) Loss of Gli3 also decreases expression of the dopamine marker TH in dorsal SVZ-derived neurons, making their fate resemble that of ventral
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
1) Researchers expressed and purified the UL35 gene product (VP26) of duck plague virus (DPV) to homogeneity.
2) Polyclonal antibodies were produced in rabbits using the purified recombinant VP26 protein.
3) Western blot analysis confirmed that the polyclonal antibodies recognized the recombinant VP26 protein.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. The goal is to identify genes associated with vulnerable plaques and rupture. Plaques from influenza-infected and drug-treated mice will also be analyzed to study effects on gene expression and plaque structure.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
This document provides a professional summary for Hariharan Ragothaman. It outlines his 8+ years of experience in software development, with 4+ years focused on Salesforce CRM. It details his technical skills and certifications in Salesforce development and administration. It also lists his work history across several roles and projects in software development, Salesforce customization, and client support.
El documento presenta la información sobre la administración educativa y la importancia y función de las matrices y comisiones en el proceso de elaboración de un código de convivencia escolar. Enumera también los 12 componentes de un código de convivencia y la función de cada uno.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
Universal and rapid salt extraction of high quality genomic dna for pcr-based...CAS0609
This document describes a simple and universal method for extracting high-quality genomic DNA from a variety of organisms including plants, fungi, insects, and shrimp. The method uses a salt-based homogenizing buffer and SDS to extract DNA from as little as 50mg of fresh tissue. The extracted DNA is of sufficient quality and quantity to be used in PCR, restriction digestion, and other molecular techniques. The method is fast, inexpensive, and does not require expensive equipment, making it suitable for laboratories with limited resources. Test results demonstrated the method successfully extracted high molecular weight DNA from many diverse organisms without modification, indicating its universal applicability.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
The document summarizes research on the Monascus genome project and related genetic studies. Key points include:
1) The Monascus genome was sequenced to 8x coverage, revealing various genomic features. A Monacolin K biosynthesis gene cluster was identified containing genes for polyketide synthases and other enzymes.
2) An efficient method for genetic transformation of Monascus pilosus was developed using aurintricarboxylic acid.
3) Disruption of the mokA gene and overexpression of the mokH transcription factor gene resulted in loss of and increased Monacolin K production, respectively.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document summarizes work done on culturing crocodile cell lines and cloning the parc gene from Pseudomonas keratitis. Primary crocodile cell lines were established from various organs and immortalized using hTERT. The parc gene was cloned from mutant and wild-type Pseudomonas strains and will be expressed and crystallized to study its role in quinolone antibiotic resistance.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
Recombinant DNA technology involves isolating and amplifying specific DNA sequences and incorporating them into vector molecules like plasmids or bacterial viruses. This recombinant DNA is then propagated in organisms like E. coli. Genetically engineered bacteria can produce important human proteins like insulin, growth hormone, and clotting factors. Restriction enzymes and DNA ligase are used to cut and join DNA fragments to create recombinant molecules.
This study demonstrates for the first time the production of recombinant human growth hormone (rhGH) from Bacillus subtilis. A hybrid gene containing the signal sequence of the B. licheniformis serine alkaline protease gene and the cDNA encoding hGH was cloned into the pMK4 plasmid and expressed under the deg promoter in B. subtilis. The rhGH produced was secreted and confirmed to have the correct mature hGH sequence. The highest rhGH concentration of 70 mg/L was obtained at 32 hours with a yield of 9 g/kg. Fermentation characteristics differed between B. subtilis containing the hybrid gene versus the plasmid alone. This expression system could be applicable for heterologous protein production from Bac
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
The document summarizes a study on gene expression during regeneration of Drosophila wing imaginal discs. Microarray analysis found over 1,000 genes differentially expressed between cut and intact discs in the first 24 hours after wounding, indicating a strong initial response to injury. Many genes involved in apoptosis, stress response, cytoskeletal activity and JNK signaling showed increased expression. Between 24-72 hours, expression returned toward normal levels as discs resumed activities interrupted by wounding. The study identifies signaling pathways and gene categories important for disc regeneration.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
This thesis examines the role of the transcription factor Gli3 in regulating neural stem cell proliferation and neuronal fate specification in the subventricular zone (SVZ). The author finds that:
1) Expression of the Gli3 repressor form is higher in dorsal SVZ neural stem cells, while expression of the activator form Gli1 is higher in ventral SVZ stem cells.
2) Conditional ablation of Gli3 in dorsal SVZ stem cells results in their progeny adopting aberrant positions deeper in the olfactory bulb compared to controls.
3) Loss of Gli3 also decreases expression of the dopamine marker TH in dorsal SVZ-derived neurons, making their fate resemble that of ventral
This study aimed to clone homologous genes of SND1, a key regulator of secondary cell wall biosynthesis, from Populus trichocarpa. The researcher amplified four SND1 homologs from P. trichocarpa cDNA using PCR. The amplified genes were cloned into E. coli and sequenced. Two colonies were found to contain the correct SND1 sequence insert, while others were false positives. Further work will express and quantify the proteins and determine their effects on other genes and phenotypes.
1) Researchers expressed and purified the UL35 gene product (VP26) of duck plague virus (DPV) to homogeneity.
2) Polyclonal antibodies were produced in rabbits using the purified recombinant VP26 protein.
3) Western blot analysis confirmed that the polyclonal antibodies recognized the recombinant VP26 protein.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. The goal is to identify genes associated with vulnerable plaques and rupture. Plaques from influenza-infected and drug-treated mice will also be analyzed to study effects on gene expression and plaque structure.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
This document provides a professional summary for Hariharan Ragothaman. It outlines his 8+ years of experience in software development, with 4+ years focused on Salesforce CRM. It details his technical skills and certifications in Salesforce development and administration. It also lists his work history across several roles and projects in software development, Salesforce customization, and client support.
El documento presenta la información sobre la administración educativa y la importancia y función de las matrices y comisiones en el proceso de elaboración de un código de convivencia escolar. Enumera también los 12 componentes de un código de convivencia y la función de cada uno.
El documento presenta información sobre varias tecnologías de comunicación como la imprenta, el código Morse, la radiodifusión pública, la televisión, las computadoras, Internet, el correo electrónico, los teléfonos móviles y las redes inalámbricas. Define cada una y explica brevemente su funcionamiento y propósito.
El libro de Levítico establece normas de santidad y pureza para la relación con Dios. Incluye prescripciones morales como juzgar con justicia y no perjudicar a otros en transacciones. También cubre el año del jubileo, cuando la tierra y propiedades deben ser devueltas, y normas para la tasación de tierras y personas consagradas a Dios.
El documento proporciona definiciones de WWW, Web 1.0, Web 2.0 y Web 3.0. WWW se refiere al sistema de hipertexto que permite acceder a sitios web a través de internet usando un navegador. Web 1.0 consistía en páginas web estáticas con contenido de solo texto. Web 2.0 introdujo blogs, wikis, redes sociales y contenido generado por usuarios. Web 3.0 se refiere a la web semántica, la nube y aplicaciones multiplataforma accesibles desde diferentes dispositivos.
This document discusses protein microarrays and their development and applications. It describes some key differences between protein and DNA microarrays, such as the challenges of amplifying and predicting protein activity and interactions due to their 3D structures. Various methods for capturing proteins on chips are presented, including different oriented immobilization techniques. Applications of protein microarrays include analyzing protein interactions, screening for drug targets, and developing techniques like self-assembly and covalent mRNA-protein fusion protein microarrays. Detection methods like fluorescence, enzymatic reactions, and mass spectrometry are also summarized.
pOnebyOne™ are efficient, accurate and flexible Bicistronic Mammalian Expression Kits that contains an Expression Cassette based in 2A sequence breakthrough technology.
Its novel (patent pending) technology allows simultaneous Expression of two Proteins from the same mRNA. Cells transfected with Bicistronic vectors ensure that if one of the Proteins is present, the other one is also present.
Bicistronic Expression vectors are supported on viral elements: the IRES or 2A sequence. IRES has been widely used. It is a relative short sequence, around 600-700 bp, although this length could be a disadvantage in viral vectors where packaging capacity is limited. IRES based Expression vectors are characterized by a non-stoichiometric production of both proteins; generally there is a lower expression of the downstream gene.
Many 2A sequences from several families of viruses have been described for producing multiple polypeptides. 2A mediated cleavage is a universal phenomenon in all eukaryotic cells. With just 20 bp in length, the 2A sequence has been used succesfully to generate multiple proteins in some biological models: plants, zebrafish, transgenic mice or eukaryotic cell lines. Vectors based on 2A produce stoichiometric proportion of both proteins.
Canvax™ offers a ready-to-clone solution of your Gene of Interest, obtained by PCR, onto a wide collection of Bicistronic vectors based on 2A sequence. You can choose among different Promoters, selection Antibiotics or Reporter Genes.
132 gene expression in atherosclerotic plaquesSHAPE Society
This document discusses microarray studies to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. It begins with background on the history and applications of DNA microarrays. Key steps discussed include probe design, sample preparation including tissue collection, labeling RNA samples, hybridizing samples to a microarray chip, scanning and analyzing image data. The document outlines creating a custom microarray based on selected genes and correlating gene expression with temperature, pH, spectroscopy and histopathology of plaques. It will also analyze gene expression in influenza-infected mice and mice where plaques are induced to rupture with drugs. Human carotid artery specimens from surgery will be analyzed from symptomatic and asymptomatic patients.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. Plaques from influenza-infected and drug-treated mice will also be analyzed to identify genes associated with plaque rupture. The goal is to better understand plaque vulnerability and identify potential drug targets.
This document discusses methods for determining cell viability. It defines viability as the capacity for replication over a given timeframe. Methods for counting viable cells include indirect dilution-based techniques where colonies are counted after culturing, and direct techniques like nalidixic acid treatment, fluorogenic dyes, and microradiography that identify viable cells without culturing. A variety of assays can also assess properties of viable cells like integrity, permeability, enzyme content, and energy status to evaluate effects on cell viability.
IRJET- Silencing of hnRNP A1 and hnRNP A2/B1 Downregulates the Expression of ...IRJET Journal
The document summarizes a study that examined the effect of silencing hnRNP A1 and hnRNP A2/B1 splice factors on the expression of CD44v6 and CD44v10 exons in glioma cells. The researchers found that knockdown of hnRNP A1 and hnRNP A2/B1 led to decreased expression of CD44v6 and CD44v10 exons based on qRT-PCR analysis. Specifically, cells with silenced splice factors had lower expression of the two exons compared to non-silenced control cells. Therefore, hnRNP A1 and hnRNP A2/B1 may positively regulate the expression of
AAV production protocol:packaging,concentration and purification-gene medissuser8cc395
1. Adeno-associated virus (AAV) is a small DNA virus that does not cause disease on its own but requires assistance from other viruses like adenovirus to replicate. Recombinant AAV (rAAV) vectors are commonly used for gene delivery by replacing the viral genome with a transgene cassette.
2. Production of rAAV involves co-transfecting three plasmids (containing the transgene, Rep/Cap genes, and adenoviral genes) into AAV-293 packaging cells. Harvested viral particles are purified using iodixanol gradient ultracentrifugation.
3. Purified rAAV is concentrated and quality is assessed by detecting the
This document provides protocols for producing recombinant adeno-associated virus (rAAV) vectors. It first describes AAV biology, noting that AAV requires helper viruses to replicate and has nonpathogenic wildtype strains. The protocol then outlines a three-plasmid system to generate rAAV using AAV-293 packaging cells. This involves transfecting cells with vectors encoding the gene of interest flanked by AAV inverted terminal repeats along with helper plasmids. Harvested rAAV is purified using iodixanol gradient ultracentrifugation and concentrated using ultrafiltration to remove contaminants. Quality control assesses the presence of three AAV capsid proteins to confirm vector purity.
Recombinant DNA technology involves joining DNA fragments from different sources to create a new DNA fragment. This involves selecting the target DNA, a cloning vector to carry the inserted DNA, DNA ligases to join the pieces, restriction enzymes to cut the DNA, and a host cell to replicate the new DNA. Common vectors include bacterial plasmids and phages that can carry inserted DNA fragments of varying sizes. The process involves cutting, joining, and replicating the DNA within the host cell. Applications include gene therapy, transgenic plants and animals, and industrial production of proteins and chemicals. Recent advances allow modifying proteins through site-directed mutagenesis.
EFFECT OF RESVERETROL ON HUMAN BREAST CANCER (MCF-7) CELL LINEAbhishek Banerjee
This document summarizes a study on the effect of resveratrol on human breast cancer MCF-7 cell lines. The study found that resveratrol inhibited the viability of MCF-7 cells in a dose-dependent manner, with an IC50 value of 125μM. Treatment with resveratrol induced apoptosis in the cells, as shown by DNA fragmentation, phosphatidylserine externalization, and morphological changes observed under electron microscopy. The apoptosis was further confirmed by the activation of apoptotic proteins like t-Bid and cleavage of α-fodrin. The study concludes that resveratrol shows promise as an anti-cancer agent for its ability to induce apoptosis in breast cancer cells.
This document summarizes a study on the effect of resveratrol on human breast cancer (MCF-7) cell lines. The study found that resveratrol inhibited the viability of MCF-7 cells in a dose-dependent manner, with an IC50 value of 125μM. Tests showed that resveratrol induced apoptosis in MCF-7 cells, as evidenced by cellular morphology changes, DNA fragmentation, and activation of apoptotic proteins like t-Bid and cleavage of α-fodrin. The study concludes that resveratrol shows potential as a promising drug for cancer treatment based on its ability to selectively induce apoptosis in cancer cells.
The document discusses developing a DNA vaccine for fish using chitosan nanoparticles to deliver plasmid DNA encoding the OMP38 gene of Vibrio anguillarum. Key points:
- Chitosan nanoparticles were developed to deliver the pVAOMP38 plasmid and protect it from nuclease degradation. Studies showed the nanoparticles maintained plasmid integrity.
- The pVAOMP38 plasmid was transfected into seabass kidney cells in vitro and shown to express.
- Fish were vaccinated by feeding with chitosan-pVAOMP38 nanoparticles, chitosan-empty vector, or chitosan alone. The fish were later challenged with V. anguillarum to evaluate vaccine efficiency.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
The document summarizes a study that examined gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). The study found that NDV infection changed the expression levels of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other cellular processes. Specifically, the study used a gene expression kit to measure changes in 21 genes related to these processes in MCF-7 cells infected with NDV. It found that NDV infection altered the expression of genes like PUMA, Bcl-2, ESR1, and MYBL2. The results provide insight into the gene regulation mechanisms by which NDV selectively kills cancer cells.
Gene expression profiling in apoptotic mcf 7 cells infected with newcastle di...Mohamed Khalid Ali Xundhur
1) The document analyzes gene expression profiling in MCF-7 breast cancer cells infected with Newcastle disease virus (NDV). It finds that NDV infection changes the expression level of many genes involved in tumor progression, cell cycle regulation, apoptosis, and other processes.
2) Specifically, it finds that NDV down-regulates expression of genes like estrogen receptor 1 (ESR1) and up-regulates genes like Bcl-2 binding component 3 (BBC3), both of which are involved in apoptosis.
3) Cell cycle analysis shows that 11% and 41% of MCF-7 cells underwent apoptosis at 3 and 6 hours post-infection respectively, indicating NDV induces apoptosis in the cancer cells
This document summarizes a presentation given by Minoru Kubo on single cell analyses for plant reprogramming studies. The presentation covered how next generation sequencing and single cell analyses have changed biological research. It discussed single cell transcriptomics for analyzing plant reprogramming at the single cell level. The goal is to identify reprogramming genes, understand cell-cell interactions during reprogramming, and validate theories of reprogramming using the moss Physcomitrella patens as a model organism. Positioning information from single cell analyses could provide new insights into cell lineages, interactions, and multicellular development.
Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA. It allows scientists to make millions to billions of copies of the target DNA sequence. Real-time quantitative PCR (qPCR) allows quantification of the amount of target DNA or RNA present. In situ hybridization is a technique that uses labeled nucleic acid probes to localize specific DNA or RNA sequences within cells in preserved tissue samples.
Currently, human papillomavirus (HPV) DNA tests validated
in large trials and epidemiological studies are the hybrid
capture second-generation (HC2) HPV DNA assay and
a variety of polymerase chain reaction (PCR) protocols employing
degenerate or consensus primers. This article describes
the currently available technology for HPV detection
and discusses novel technologies and their potential for
large-scale screening. Ideally, an HPV test should allow detection
of multiple HPV types, identify individual types, and
provide quantitative information about the viral load of each
individual type found. Moreover, it should be easy to perform,
be highly reproducible, with a high specificity and
sensitivity, and amenable for high throughput analysis and
automation. Because we do not yet fully understand the true
value of viral load and the biological relevance of the different
HPV types, any HPV test should be able to detect the
clinically relevant high-risk types with a sufficient sensitivity
of at least 10 000 genome copies per sample. To validate the
different current and future test systems and to compare
inter-laboratory performance we urgently need reference
samples, validated reagents, and standardized protocols.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
This document provides the timetable and protocols for a practical course on making and analyzing tRNA synthetases in vivo and using cell-free protein synthesis. Over two weeks, students will perform site-directed mutagenesis to produce mutant aminoacyl-tRNA synthetases, express their proteins in E. coli cells and purify the proteins, and use cell-free synthesis to attempt incorporating a phosphotyrosine analogue into a target protein using their mutant synthetases and suppressor tRNA. The document outlines the experimental steps, including mutagenesis, transformation, plasmid preparation, sequencing, protein expression and purification, cell-free reaction set up, and analysis by SDS-PAGE. Safety procedures are also described to handle
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
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