This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Recombinant baculoviruses are widely used to
express heterologous genes in cultured insect cells
and insect larvae. For large-scale applications, the
baculovirus expression vector system (BEVS) is particularly
advantageous.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE Dextran, calcium phosphate, and electroporation. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity. In addition, this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections). High throughput screening assay has also shown good efficiency in some in vivo models.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
Recombinant baculoviruses are widely used to
express heterologous genes in cultured insect cells
and insect larvae. For large-scale applications, the
baculovirus expression vector system (BEVS) is particularly
advantageous.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE Dextran, calcium phosphate, and electroporation. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity. In addition, this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections). High throughput screening assay has also shown good efficiency in some in vivo models.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
Hi, I am RAFi ,student of Genetic Engineering and Biotechnology , Jashore university of science & Technology. It is my first uploading slide in slideshare.I am so glad for doing this work.
for cloning and expression of exogenous gene or gene throthrough vector it must be introduced into the host cell through transformation , ,transduction, electroporation gene gun etc.
A detailed explanation of cloning strategies which involves isolation of DNA fragments from the sample and introduction in to a vector with restriction enzymes and introduced in to host by different methods and finally screening of the host cells with the recombinants based on protein,nucleicacid and antibiotic assays
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
1. Transformation and Transfection
into Prokaryotic and Eukaryotic Cells
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India
2. Transformation
• Transformation: the genetic alteration of a cell
resulting from the introduction, uptake and expression
of foreign genetic material (DNA) in molecular biology
• This can be done to Bacteria, Fungi, Plants, and Animal
cells
3. Transformation - History
• 1928 - Frederick Griffith transforms non-pathogenic
pneumococcus bacteria into a virulent variety by mixing
them with heat-killed pathogenic bacteria.
• Transformation principle was demonstrated in 1944 by
Oswald Avery, Colin MacLeod, and Maclyn McCarty,
who showed gene transfer in Streptococcus pneumoniae
was pure DNA.
• Avery, Macleod and McCarty call the uptake and
incorporation of DNA by bacteria transformation.
4. Transformation - Mechanisms
• Bacteria
– transformation refers to a genetic change brought
about by picking up naked strands of DNA and
expressing it.
– Competence refers to the state of being able to take
up DNA.
– Two different forms of competence should be
distinguished, natural and artificial.
5. Transformation - Mechanisms
• Bacteria - Natural competence
– Some bacteria (around 1% of all species) are naturally
capable of taking up DNA. Such species carry sets of genes
specifying machinery for bringing DNA across the cell's
membrane or membranes.
– The evolutionary function of these genes is controversial.
– Although most textbooks and researchers have assumed
that cells take up DNA to acquire new versions of genes, a
simpler explanation that fits most of the observations is that
cells take up DNA mainly as a source of nucleotides, which
can be used directly or broken down and used for other
purposes
6. Transformation - Mechanisms
• Bacteria - Artificial competence
– Artificial competence is not encoded in the cell's genes.
– It is induced by laboratory procedures in which cells are
passively made permeable to DNA, using conditions that do
not normally occur in nature.
– These procedures are comparatively easy and simple, and
are widely used to genetically engineer bacteria.
– Artificially competent cells of standard bacterial strains may
also be purchased frozen, ready to use.
– Common Strain of E. coli - DH5α (alpha)
7. Prokaryotic Transformation
First, the DNA…
DNA is most easily taken up if it is in plasmid form (as opposed to
linear form… although certain cells can take up linear DNA) If the
plasmids are nicked, or have been re-ligated, this can lower
transformation efficiency– supercoiled DNA gives the highest
transformation efficiency.
Generally, the plasmid will have an antibiotic-resistance marker (i.e.
tetracycline, kanamycin, or ampicilin, which stop bacterial growth
through different means) so that the cells that were successfully
transformed can be identified.
8. Then, the cells…
Competent: able to take up DNA. Although some bacteria are
naturally competent, most have to be made competent in the
lab. This is known as “artificial competence.”
We can get the cells already competent (ordered from a
company) or we can make cells competent in the lab.
Two common ways to achieve prokaryotic cell
competence are:
1. Electroporation (also works for eukaryotes)
2. Using calcium chloride CaCl2
CaCl2 CaCl2
CaCl2
CaCl2
9. Transformation - Mechanisms
• Bacteria - Artificial competence - Temperature
– Chilling cells in the presence of divalent cations such as Ca2+
(in CaCl2) prepares the cell walls to become permeable to
plasmid DNA.
– Cells are incubated with the DNA and then briefly heat
shocked (42o
C for 30-120 seconds), which causes the DNA to
enter the cell.
– This method works well for circular plasmid DNAs but not
for linear molecules such as fragments of chromosomal
DNA.
– An excellent preparation of competent cells will give ~108
colonies per μg of plasmid. A poor preparation will be about
104
/μg or less. Good non-commercial preps should give 105
to
106
transformants per microgram of plasmid.
10. The CaCl2 method
This method also alters the permeability of the cell membrane:
• Ca2+
interacts with the negatively charged phospholipid heads of the
cell membrane, creating an electrostatically neutral situation.
• Lowering the temperature stabilizes the membrane, making the
negatively charged phosphates easier to shield.
• Then a heat shock creates a temperature imbalance and thus a
current, which helps get the DNA into the cell.
11. Transformation - Mechanisms
A plasmid again?
• A plasmid DNA molecule contains sequences allowing it to be
replicated in the cell independently of the chromosome.
• Plasmids used in experiments will usually also contain an
antibiotic resistance gene which is placed in a bacterial strain
that has no antibiotic resistance.
• Therefore, only transformed bacteria will grow on a media
containing the antibiotic.
12. Transformation - Mechanisms
• Bacteria - Artificial competence –
Electroporation
– Electroporation is another way to make holes in cells, by
briefly shocking them with an electric field of 100-200 V/cm.
– Now plasmid DNA can enter the cell through these holes.
– Natural membrane-repair mechanisms will close these holes
afterwards.
13. Electroporation!
The general idea behind electroporation is that by applying a
short electrical pulse to the cells, we can alter membrane
conductivity and permeability. It is more effective than the CaCl2
method (chemical competence).
DNA is a negatively
charged molecule due
to phosphate groups (in
its “backbone”).
electroporated –
hydrophilic pore
normal
Polar molecules
don’t normally
cross the cell
membrane easily
because the
inside is
hydrophobic.
But
electroporation
makes pores in
the membrane
that are
hydrophilic,
enabling DNA to
pass through.
14. 1. Inoculate a colony into ~50 ml (no salt) LB and grow at 37°C
overnight.
2. Add ~25 ml culture medium into 1 L LB medium.
3. Grow the cells at 37°C in a shaking incubator.
4. Grow cells to an A600 of ~0.6-0.7. This represents the bacteria’s
log-phase growth. Why log phase? Cells in this phase are
growing rapidly, and are healthy and uniform. (Also keep in
mind that since they divide so rapidly, you should work at a
decent pace.)
5. Pour ~250 mL into a tube and spin down in a centrifuge at 4°C.
6. Remove supernatant and resuspend cells in dH2O.
7.Repeat centrifugation / removal of
supernatant several times.
8.Resuspend in 10% glycerol and keep
in freezer until ready to use.
To make electrocompetent cells:
If wastes were removed and nutrients
were supplied infinitely, the bacteria
would keep growing. But because that’s
not the case, at stationary phase, the rate
of cell growth equals the rate of cell
death.
15. To electroporate:
1. Keep cells cold (on ice)!
2. Prepare the DNA you want to put into the cells (i.e. dilute it. Usually you don’t
need a very high DNA concentration).
3. Pipette some (~100 µL) cells and DNA (~1 µL?)
into a cuvette.
4. After making sure the settings on the
electroporator are correct, put the cuvette in
and press the button. Your settings should
maximize the number of transformed bacteria
while also keeping as many alive as possible.
5. Within 30 seconds of electroporation, pipette
about half a mL of SOC (recovery medium). SOC is basically a bunch of salts,
glucose, amino acids (tryptone) and some yeast extract. Mix.
6. Let the cells recover at 37°C in a shaking incubator for about an hour. Shocking
them stresses them out.
7. Plate the cells and let them grow.
16. Arcing…
• If you see or hear a spark coming from the cuvette, then the cells are
dead! Repeat that sample.
• Things that can cause arcing:
– excess water on cuvette outside
– human skin oil on cuvette outside
– too high salt concentration in DNA sample (try diluting DNA.)
17. Transformation - Mechanisms
Bacteria - Artificial competence – Lipofection
Lipofection (or liposome transfection) is a technique used to
inject genetic material into a cell by means of liposomes
which are vesicles that can easily merge with the cell
membrane since they are both made of a phospholipid
bilayer.
The vescicle fuses with the cell membrane (similar to how
two oil spots at the top of a broth will fuse) and the
contents of the vesicle & the cell are combined.
18. Transfection
• Transfection: the introduction of foreign material into
eukaryotic cells.
• This typically involves opening transient pores or 'holes' in the
cell plasma membrane, to allow uptake of material.
19. Other biochemical methods
Lipofection
•Uses cationic liposomes that
form a complex with DNA
•DNA is not encapsulated within
the liposomes, but bound to the
outside
Dendrimers
•Dendrimers are highly
branched molecules that form a
complex with DNA
Once DNA has formed a
complex with these
molecules, endocytosis
allows the complex to enter
the cells
20. Lipofection
The cells should be 75% confluent at the time oflipofection.
For each dish of cultured cells to be transfected, dilute 1-10 µg of
plasmid DNA into 100µl of steriledeionized H2O (if using Lipofectin) or
20 mM sodium citrate containing 150 mM NaCl (pH 5.5) (if using
Transfectam) in a polystyrene or polypropylene test tube. In a
separate tube, dilute 2-50 µl of the lipid solution to a final volume of
100 µl with sterile deionized H2O or 300 mM NaCl.
When transfecting with Lipofectin, use polystyrene test tubes; do not
use polypropylene tubes, because the cationic lipid DOTMA can bind
nonspecifically topolypropylene
Incubate the tubes for 10 minutesat room temperature
Add the lipid solution to the DNA,and mix the solution bypipetting up
and down several times. Incubate the mixture for 10 minutes at room
temperature.
21. Lipofection (cont’d)
• While the DNA-lipid solution is incubating, wash the cells to be
transfected three times with serum-free medium. After the third
rinse, add 0.5 ml of serum-free medium to each 60-mm dish and
return the washed cells to a 37°C humidified incubator with an
atmosphere of 5-7% CO2.
It is very important to rinse the cells free of serum before the
addition of the lipid-DNAliposomes.
• After the DNA-lipid solution has incubatedfor 10 minutes,add 900
µl of serum-free medium to each tube. Mix the solution by
pipetting up and down several times. Incubate the tubes for 10
minutes at room temperature.
• Transfer each tube of DNA-lipid-medium solution to a 60-mmdish
of cells. Incubate the cells for 1-24 hours at 37°C in a humidified
incubator with an atmosphere of 5-7% CO2.
• Afterthe cells have been exposed to the DNA for the appropriate
time, wash them three times with serum-free medium. Feed the
cellswith complete medium and return them to the incubator.
• If the objective is stable transformation of the cells, select for
those cells after 24-72 hours
22. Nucleofection
• Transfects DNA directly into the nucleus without requiring dividing cells
or viral vectors
• Uses a combination of electrical parameters and cell-type specific
reagents
• Provides the ability to transfect even non-dividing cells, such as neuron
and resting blood cells
• Optimal nucleofection conditions depend on the individual cell type, not
the substrate being transfected
The future of electroporation?
Nucleofection basics:
0.5 - 1.5 x 106
cells
2-5 µg highly purified plasmid DNA (in max. 5 µl H2O or TE)
100 µl Nucleofector Solution (cell-type specific)
Perform each sample separately to avoid storing the cells longer than 15 min
in Nucleofector Solution.
Cells should be nucleofected at 70-80% confluency.
23. Transfection Mechanisms
Yeasts and Fungi
• These methods (and more) are currently known to transform
yeasts:
• Two-hybrid System Protocol:
– The two-hybrid system involve the use of two different plasmids in a
single yeast cell.
– One plasmid contains a cloned gene or DNA sequence of interest while the
other plasmid contains a library of genomic or cDNA.
• Frozen Yeast Protocol:
– Frozen yeast cells that are competent for transformation after thawing.
• Gene Gun Transformation:
– Gold or tungsten nanoparticles can be shot at fungal cells growing on PDA,
transforming them.
• Protoplast Transformation:
– Fungal spores can be turned into protoplasts which can then be soaked in
DNA solution and transformed.
24. Transfection Mechanisms
• Plants - A number of mechanisms are available to transfer DNA into an
organism, these include:
– Agrobacterium is a genus of Gram-negative bacteria that
uses horizontal gene transfer to cause tumors in plants.
Agrobacterium tumefaciens is the most commonly studied
species in this genus.
– Horizontal gene transfer (HGT), also Lateral gene transfer
(LGT), is any process in which an organism incorporates
genetic material from another organism without being the
offspring of that organism.
– By contrast, vertical transfer occurs when an organism
receives genetic material from its ancestor, e.g. its parent or
a species from which it evolved.
– Most thinking in genetics has focused upon vertical transfer,
but there is a growing awareness that horizontal gene
transfer is a highly significant phenomenon, and amongst
single-celled organisms perhaps the dominant form of
genetic transfer.
– Artificial horizontal gene transfer is a form of
genetic engineering.
25. Transfection Mechanisms
• Plants - A number of mechanisms are available to transfer DNA into
an organism, these include:
– Agrobacterium mediated transformation is the easiest and
most simple plant transformation.
– Plant tissue (often leaves) are cut in small pieces, eg.
10x10mm, and soaked for 10 minutes in a fluid containing
suspended Agrobacterium.
– Some cells along the cut will be transformed by the
bacterium, that inserts its DNA into the cell.
– Placed on selectable rooting and shooting media, the
plants will regrow.
– Some plants species can be transformed just by dipping
the flowers into suspension of Agrobacteria and then
planting the seeds in a selective medium.
– Unfortunately, many plants are not transformable by this
method.
26. Transfection Mechanisms
• Plants
– Electroporation: make holes in cell walls using electricity,
that allows DNA to enter.
– Viral transformation: Package your genetic material into a
suitable plant virus and then use the modified virus for
infection of the plant.
– Genomes of most plant viruses consist of single stranded
RNA which replicates in the cytoplasm of infected cell.
– So this method is not a real transformation (why?) … since
the inserted genes never reach the nucleus of the cell and do
not integrate into the host genome.
– The progeny of the infected plants is virus free and also free
of the inserted gene
27. Transfection Mechanisms
• Plants
Particle bombardment (gene gun):
– Coat small gold or tungsten particles with DNA and shoot
them into young plant cells or plant embryos.
– Some genetic material will stay in the cells and transform
them.
– This method also allows transformation of plant plastids.
• The transformation efficiency is lower than in Agrobacterial
mediated transformation, but most plants can be transformed with
this method.
28. Transfection Mechanisms
• More on the “gene gun”
• The target of a gene gun is often a callus of undifferentiated
plant cells growing on gel medium in a petri dish.
• After the gold particles have impacted the dish, the gel and
callus are largely disrupted. However, some cells were not
obliterated in the impact, and have successfully enveloped a
DNA coated tungsten particle, whose DNA eventually migrates
to and integrates into a plant chromosome.
• Cells from the entire petri dish can be re-collected and selected
for successful integration and expression of new DNA using
modern biochemical techniques
• Selected single cells from the callus can be treated with a series
of plant hormones, such as auxins and gibberellins, and each
may divide and differentiate into the organized, specialized,
tissue cells of an entire plant. This capability of total re-
generation is called totipotency. The new plant that originated
from a successfully shot cell may have new genetic (heritable)
traits.
29. Gene Gun (biolistic particle delivery)
• Uses compressed gas to deliver DNA-coated heavy metal particles
• Able to transform almost any type of cell
• Mostly used for plant cells
• Can inject dyes, plastids, vaccines, and other substances
• More suited to tissues than small cells or cultures, as the high
velocity particles have a high chance of rupturing cells (pit effect)
30. Transfection Mechanisms
• Gene gun with Humans and Animals
• Gene guns have also been used to deliver DNA vaccines to
experimental animals. Theoretically, it may be used in humans
as well.
• The delivery of plasmids into rat neurons through the use of a
gene gun is also used as a pharmacological precursor in
studying the effects of neurodegenerative diseases such as
Alzheimer's Disease.
• The Gene gun technique is also popularly used in Edible vaccine
production technique, where the nano gold particles coated
with plant gene under the high vacuum pressurized chamber is
transformed into suitable plant tissues.
31. Calculating Transformation Efficiency
(Don’t you want to see how effective your hard work was?)
Transformation efficiency (transformants/µg)
is calculated as follows:
# colonies on plate/ng of DNA plated X 1000 ng/µg
Things that affect transformation efficiency:
• Actual DNA Concentration
• Forms of DNA - Linear and single-stranded DNA transforms <1%
as efficiently as supercoiled DNA.
• Purity of DNA- DNA can be contaminated with salts. Also, ligase
can interfere with transformation. You can heat-inactivate the
ligase before the transformation. You can also column-purify
your DNA.
• Freeze/Thawing of Cells - Cells that are refrozen will lose
activity, typically at least two-fold.
32. Transfection Mechanisms
• Animals
– Microinjection: use a thin needle and inject the DNA directly
in the core of embryonic cells.
– Viral transformation: Package genetic material into a virus,
which delivers the genetic material to target host cells.
33. Microinjection
• Single cell at a time
• Requires major precision, time, and labor (laborious)
• DNA is inserted directly into nucleus (high success factor)
CELL PREPERATION:
1. Plate cells on a glass coverslip.
2. For a good injection, a 60-80% cell confluence at the day of injection is
required.
3. The day of the experiment transfer each coverslip in a 6 cm diameter
plate with 5 ml of medium/plate
DNA PREP
1. Dilute the DNA in ddH2O to a final concentration of 20-150 ng/µl
2. Centrifuge 15 min. at 13,000 rpm RT and transfer the supernatant in a
new clean eppendorf tube.
3. You can mix different DNA but the final concentration has to be 150
ng/µl total max. Alternatively IgGs can be mixed to the DNA in order
to use them as microinjection efficiency marker.
4. Inject the sample into target cell nuclei
34. A Quick Note:
Generally, it is good practice to do a control transformation
(with water) just to aid any future necessary troubleshooting. If
you get colonies on the control plate, something definitely
went wrong with your transformation.
?
35. Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
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