Transformation and transfection

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

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

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.

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.

• 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

• 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)

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.

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

• 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.

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.

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.

• 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.

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.

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.

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.

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.)

 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.

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.

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

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.

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

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.

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.

• 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.

• 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.

• 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

• 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.

• 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.

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)

• 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.

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.

• 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.

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

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.
?

Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
Questions???

Transformation and transfection

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