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Overview
Common Transfection Methods
Reagent-Based Methods
Lipids
Calcium phosphate
Cationic polymers
DEAE-dextran
Activated dendrimers
Magnetic beads
Instrument-Based Methods
Electroporation
Biolistic technology
Microinjection
Laserfection/optoinjection
Virus-Based Methods
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Terminology
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Factors Affecting Transfection
Host Cell
Cell health
Cell culture
Genetic Material
DNA quality and quantity
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Terminology
Transfection: Introduction of foreign DNA into the nucleus of eukaryotic cells.
Cells that have incorporated the foreign DNA are called transfectants.
Stable transfectants: Cells that have integrated foreign DNA in their genome.
Transient transfectants: Foreign DNA does not integrate in the genome but
genes are expressed for a limited time (2496 hours).
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Host Cell
Cell health
Cell culture
Genetic Material
DNA quality and quantity
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Host Cell
Cell Health

Cells should be grown in appropriate medium with all necessary factors

Cultures must be free of contamination

Fresh medium must be used if it contains chemically unstable components, such as thiamine

Cells should be incubated at 37C with CO
2
supplied at the correct percentage
(510%) and 100% relative humidity

Cells should be maintained in log phase growth
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Cell Culture
Conuency and Growth Phase

Cells should be transfected at 4080% conuency (cell type dependent)
Too few cells cause cell cultures to grow poorly without cell-to-cell contact
Too many cells result in contact inhibition, making cells resistant to uptake of DNA
and other macromolecules

Actively dividing cells take up DNA better than quiescent cells (breakdown and perforation
of the nuclear membrane during mitosis enable nuclear delivery)
Number of Passages for Primary Cells

The number of passages should be low (<50)

The number of passages for cells used in a variety of experiments should be consistent

Cell characteristics can change over time with immortalized cell lines and cells may not
respond to the same transfection conditions

Cells may not respond to the same transfection conditions after repeated passages
Host Cell Cell Culture
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DNA Quality and Quantity

Use high-quality plasmid DNA that is free of proteins, RNA, and chemicals for transfections;
endotoxin removal should be part of the preparation procedure

Typically, DNA is suspended in sterile water or TE buffer to a nal concentration of 0.21 mg/ml

The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA,
transfection reagent/method, target cell line, and number of cells
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Tissue culture
Count cells
Resuspend cells in
electroporation buffer
Add nucleic acid
Transfect cells
Plate cells
Analysis Protein expression Gene expression
Reporter gene activity Western blot analysis Microscopy Flow cytometry Real-time qPCR
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Reagent-Based Methods
Lipids
Calcium phosphate
Cationic polymers
DEAE-dextran
Activated dendrimers
Magnetic beads
Instrument-Based Methods
Electroporation
Biolistic technology
Microinjection
Laserfection/optoinjection
Virus-Based Methods
Common
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Methods
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Cationic Lipids: Liposomes/Lipoplexes

Cationic lipids are amphiphilic molecules that have a positively charged polar head group linked,
via an anchor, to an apolar hydrophobic domain generally comprising two alkyl chains

Structural variations in the hydrophobic domain of cationic lipids include the length and the degree of
non-saturation of the alkyl chains

Electrostatic interactions between the positive charges of the cationic lipid head groups and the
negatively charged phosphates of the DNA backbone are the main forces that allow DNA to spontaneously
associate with cationic lipids
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Lipid-Mediated
Gene Delivery
Lipid-mediated gene delivery is also referred to as lipofection, or
liposome-based gene transfection. It uses lipids to cause a cell
to absorb exogenous DNA.
Transfer of genetic material into the cell takes place via
liposomes, which are vesicles that can merge with the cell
membrane since they are both made of a phospholipid bilayer.
Lipids Reagent-Based Methods
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Methods
Nucleic acid
Transfection
Endocytosis
Cationic lipid
Liposome
Analysis
Complexing
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Solution A Solution B
Plated cells Aspirate medium from cells Incubate overnight and assay
0.251 g plasmid DNA
per 50 l serum-free medium
Mix equal volumes of TransFectin
and DNA solutions
Incubate 20 min to form DNA-liposome complexes
Mix; add DNA-liposome complexes directly to cells
(100 l/24-well plate)
24 l Transfectin

lipid reagent
per 50 l serum-free medium
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Pros and Cons
Advantages of Lipids

Deliver nucleic acids to cells in a culture dish with high efciency

Easy to use, minimal steps required; adaptable to high-throughput systems

Using a highly active lipid will reduce the cost of lipid and nucleic acid, and achieve effective results
Disadvantage of Lipids

Not applicable to all cell types
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Calcium Phosphate
The protocol involves mixing DNA with calcium chloride,
adding this in a controlled manner to a buffered saline/
phosphate solution, and allowing the mixture to incubate
at room temperature.
This step generates a precipitate that is dispersed onto the
cultured cells. The precipitate is taken up by the cells via
endocytosis or phagocytosis.
Calcium Phosphate Reagent-Based Methods
Ca
++
Ca
++
Ca
++
Ca
++
Calcium
phosphate
Ca
++
Ca
++
DNA
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Calcium Phosphate Reagent-Based Methods
Method Overview
Solution A: DNA in calcium solution
Solution B: 2x Hanks buffered saline solution
1 Add solution A to solution B while vortexing.
2 Incubate 2030 min. Apply the solution to a subconuent cell culture.
3 Incubate 212 hr. Replace the solution with complete growth medium.
4 Assay for transient gene expression or begin selection for
stable transformation time.
Common
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Solution A
Step 1
Step 2
Step 3
Step 4
Solution B
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Pros and Cons
Advantages of Calcium Phosphate

Inexpensive

High efciency (cell type dependent)

Can be applied to a wide range of cell types

Can be used for transient and stable transfection
Disadvantages of Calcium Phosphate

Reagent consistency is critical for reproducibility

Small pH changes (0.1) can compromise transformation efciency

Size and quality of the precipitate are crucial to the success of transfection

Calcium phosphate precipitation does not work in RPMI, due to the high
concentration of phosphate within the medium
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Cationic Polymers
Cationic polymers differ from cationic lipids in that they do not contain a hydrophobic
moiety and are completely soluble in water. Given their polymeric nature, cationic polymers
can be synthesized in different lengths, with different geometry (linear versus branched).
The most striking difference between cationic lipids and cationic polymers is the ability of
the cationic polymers to more efciently condense DNA.
There are three general types of cationic polymers used in transfections:

Linear (histone, spermine, and polylysine)

Branched

Spherical
Cationic polymers include polyethyleneimine (PEI) and dendrimers.
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Headline
CH
2
CH
3
O CH
2
H CH
2
N
+
CH
2
CH
3
Diethylaminoethyl
DEAE-Dextran Reagent-Based Methods
Dextran
DEAE-Dextran
DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic
acids. The positively charged DNA:polymer complex comes into close association with the
negatively charged cell membrane. DNA:polymer complex uptake into the cell is presumed
to occur via endocytosis or macropinocytosis.
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Transfection Methods DEAE-Dextran Reagent-Based Methods
Method Overview
Solution A: DNA (~15 g/ml) diluted into 2 ml of growth
medium with serum containing chloroquine
Solution B: DEAE-dextran solution (~50500 g/ml)
Solution C: ~5 ml of DMSO
Solution D: Complete growth medium
1 Add solution A to solution B, then mix gently.
2 Aspirate cell medium and apply the mixed A and B solutions to the
subconuent cell culture. Incubate the DNA mixture for ~4 hr.
3 Aspirate supernatant.
4 Add solution C to induce DNA uptake.
5 Remove DMSO and replace with complete growth medium; assay for
transient gene expression.
Common
Transfection
Methods
Solution A
Step 1
Step 2
Step 3
Step 4
Step 5
Solution B
Solution C
Solution D
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Pros and Cons
Advantages of DEAE-Dextran

Inexpensive

Easy to perform and quick

Can be applied to a wide range of cell types
Disadvantages of DEAE-Dextran

High concentrations of DEAE-dextran can be toxic to cells

Transfection efciencies will vary with cell type

Can be used only for transient transfection

Typically produces less than 10% delivery in primary cells
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Activated
Dendrimers Structure
Positively charged amino groups (termini) on the surface of
the dendrimer molecule interact with the negatively charged
phosphate groups of the DNA molecule to form a DNA-
dendrimer complex.
The DNA-dendrimer complex has an overall positive net charge
and can bind to negatively charged surface molecules on the
membrane of eukaryotic cells. Complexes bound to the cell
surface are taken into the cell by nonspecic endocytosis. Once
inside the cell, the complexes are transported to the endosomes.
DNA is protected from degradation by endosomal nucleases by
being highly condensed within the DNA-dendrimer complex.
Amino groups on the dendrimers that are unprotonated at
neutral pH can become protonated in the acidic environment of
the endosome. This leads to buffering of the endosome, which
inhibits pH-dependent endosomal nucleases.
Activated Dendrimers Reagent-Based Methods
Common
Transfection
Methods
Generation
numbers
Termini
Branching
points
Dendrimer
Core
G4
G3
G2
G1
G0
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Magnet-Mediated Transfection
Magnet-mediated transfection uses magnetic force to deliver DNA into target cells.
Nucleic acids are rst associated with magnetic nanoparticles. Then, application
of magnetic force drives the nucleic acidparticle complexes toward and into the
target cells, where the cargo is released.
Magnetic Beads Reagent-Based Methods
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Magnetic Beads Reagent-Based Methods
Method Overview
1 Dilute nucleic acid in medium.
2 Add magnetic nanoparticle.
3 Incubate 1020 min.
4 Add medium to adherent cells (24 x 10
5
cells).
5 Add nucleic acid/nanoparticle solution.
6 Place culture plate on magnet plate.
7 Incubate 15 min.
8 Remove magnet plate.
Positively charged
magnetic particles
Negatively
charged DNA
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Pros and Cons
Advantages of Magnetic Beads

Rapid

Increased transfection efciency by the directed transport, especially for low amounts
of nucleic acids

High transfection rates for adherent mammalian cell lines and primary cell cultures
(suspension cells and cells from other organisms also successfully transfected but
need to be immobilized)

Mild treatment of cells

Can also be performed in the presence of serum
Disadvantages of Magnetic Beads

Relatively new method

Requires adherent cells; suspension cells need to be immobilized or centrifuged
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+
+
+
+
Field Removed Membrane Heals
Field Applied Pathways Form
Field Applied Ions Move
Ions Gene
Cell
membrane
Before Field Applied
Electroporation Instrument-Based Methods
How Electroporation Works
1 Electroporation exposes a cell to a high-intensity electric eld that temporarily
destabilizes the membrane.
2 During this time the membrane is highly permeable to exogenous molecules
present in the surrounding media.
3 DNA then moves into the cell through these holes.
4 When the eld is turned off, the pores in the membrane reseal, enclosing the
DNA inside.
Step 1
Step 2
Step 3
Step 4
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V
o
l
t
a
g
e
,

V
Time, ms Time, ms
t
V
0
V
0
V
t
V
0
e
t
V
o
l
t
a
g
e
,

V
Type of Electrical Pulse the Waveform
The most common electrical elds are exponential and square waveforms. The waveform has a signicant
effect on the transfection efciency for different cell types. Both exponential-decay and square-wave pulses
have been used very effectively for electroporation.
Exponential Waveform

Capacitors charged to a set voltage

Set voltage is released from the selected capacitor
and decays rapidly (exponentially) over time
Electroporation Instrument-Based Methods
Square Waveform

Determined by pulse duration and/or number of pulses

Several short pulses may be more benecial than one long pulse
V
o
l
t
a
g
e
,

V
Time, ms Time, ms
t
V
0
V
0
V
t
V
0
e
t
V
o
l
t
a
g
e
,

V
Common
Transfection
Methods
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Pros and Cons
Advantages of Electroporation

Nonchemical method that doesnt seem to alter the
biological structure or function of the target cells

Easy to perform

High efciency

Can be applied to a wide range of cell types
Disadvantage of Electroporation

Cell mortality (if using suboptimal conditions)
Electroporation Instrument-Based Methods
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Biolistic Particle Delivery
Biolistic transformation is the delivery of nucleic acids into cells via high velocity nucleic acidcoated microparticles.
Biolistic Technology Instrument-Based Methods
Helios

Gene Gun

For in situ, in vivo, and in vitro transformations

Applications for animals, plants, cell culture, nematodes,
yeast, and bacteria

Pressure range 100600 psi enables ne-tuning of penetration

Highly portable can be used in the eld

Small target area for accurate targeting
PDS-1000/He

Biolistic Particle Delivery System

For in vitro, ex vivo, and in vivo (for some plants and microbes)
transformations

Applications for animal cell and organ cultures, plant cell cultures
and explants, pollen, insects, algae, fungi, and bacteria

Pressure range 4502,200 psi gives exibility and penetration
ideal for plant applications

Large target area more cells can be transformed
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Instrument-Based Methods Biolistic Technology
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6
Helios

Gene Gun Process Overview


1 Precipitate DNA onto gold particles.
2 Load DNA/gold into tubing.
3 Rotate tubing to coat DNA gold over inside surface.
4 Cut tubing into cartridges.
5 Load cartridges into gene gun.
6 Deliver DNA into target cells.
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PDS-1000/He

System Process Overview


Gas acceleration tube
Rupture disk
DNA-coated microcarriers
Target shelf
Target cells
Stopping screen
Macrocarrier
Microcarrier launch assembly
Instrument-Based Methods Biolistic Technology
1 DNA-coated gold particles (microcarrier) are spread over the central area of a thin plastic disk (macrocarrier).
2 Disk loaded with the DNA-gold particles is placed into a holder inside the PDS-1000/He system.
3 The system uses high-pressure helium, released by a rupture disk, and partial vacuum to propel the macrocarrier
loaded with microcarrier toward the target cells.
4 Macrocarrier is stopped after a short distance by a stopping screen.
5 DNA-coated gold particles continue travelling toward the target to penetrate the cells.
6 Sample chamber is subjected to partial vacuum, from 15 to 29 inches of mercury, depending on the target cells.
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Helios

Gene Gun vs. PDS-1000/He

System
Factors Affecting Transformation Helios Gene Gun System PDS-1000/He System PDS-1000/He

System With Hepta

Adaptor
Experimental conditions In situ, in vitro, in vivo, ex vivo In vitro, ex vivo, in vivo (plants) In vitro, ex vivo, in vivo (plants)
Target area Small (2 cm
2
) Large (40 cm
2
) Largest (~75 cm
2
)
Pressure range 100600 psi 4502,200, psi 4502,200 psi, reduced by 7-way spread of helium
Target type Animals: Any tissue exposed Animals: Cell and organ culture Animals: Cell and organ culture
to barrel (skin, organs); cell,
explant, and organ culture
Plants: Field and greenhouse
use, plant cell culture, explants
Yeast, bacteria, other microbes
Plants: Small intact plants, Plants: Cells with thin cell walls
plant cell culture, explants
Yeast, bacteria, other microbes
Organelles (chloroplasts,
mitochondria, etc.)
Yeast, bacteria, other microbes
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Pros and Cons
Advantages of Biolistic Technology

Simple, rapid, versatile technique

Targeted intracellular gene delivery

Cell type independent

Uses small amounts of DNA

Delivers single or multiple genes

No carrier DNA needed

Can deliver large DNA fragments

No extraneous genes or proteins delivered

Requires little manipulation of cells

High reproducibility
Disadvantages of Biolistic Technology

Generally lower efciency compared to electroporation or viral- or lipid-mediated transfection

Limited bacterial transfection data

Preparation of microparticles

Instrument cost

Requires purchase agreement
Instrument-Based Methods Biolistic Technology
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Microinjection

Direct injection of naked DNA

Laborious (one cell at a time)

Technically demanding and costly

Can be used for many animals
Fertilized egg, 100 m
Injecting pipet Holding pipet
Transgene
Microinjection Instrument-Based Methods
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Impermeant
nanoparticles
Intact cell 532 nm laser beam Transiently permeabilized Intact cell
Laserfection/
Optoinjection
Laserfection/Optoinjection

This procedure uses laser light to transiently permeabilize a large number of cells in a very short time

Various substances can be efciently optoinjected, including ions, small molecules, dextrans, short interfering
RNAs (siRNAs), plasmids, proteins, and semiconductor nanocrystals, into numerous cell types

Advantages: very efcient; works with many cell types; fewer cell manipulations needed

Disadvantages: requires the cells to be attached; expensive laser-based equipment needed
Instrument-Based Methods
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Transfection Methods
Viral Vectors
Retroviruses

Murine leukemia virus (MuLV)

Human immunodeciency virus (HIV)

Human T-cell lymphotropic virus (HTLV)
DNA Viruses

Adenovirus

Adeno-associated virus (AAV)

Herpes simplex virus (HSV)
Retroviruses a class of viruses that can create double-stranded DNA copies of their RNA genomes;
these copies can be integrated into the chromosomes of host cells. HIV is a retrovirus.
Adenoviruses a class of viruses with double-stranded DNA genomes that cause respiratory, intestinal,
and eye infections in humans. The virus that causes the common cold is an adenovirus.
Adeno-associated viruses a class of small, single-stranded DNA viruses that can insert their genetic
material at a specic site on chromosome 19.
Herpes simplex viruses a class of double-stranded DNA viruses that infect a particular cell type,
neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.
Virus-Based Methods
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Viral Vector DNA Insert Size Maximum Titer Cell Type Expression Pitfalls
Retroviral 8 kb 1 x 10
9
Dividing cells Stable Random insertion site
Lentivirus 9 kb 1 x 10
9
Dividing cells Stable Random insertion site
Nondividing cells
Adenovirus 8 kb 1 x 10
13
Dividing cells Transient Highly immunogenic
Nondividing cells
Adeno-associated virus 5 kb 1 x 10
11
Dividing cells Stable, site-specic Requires helper virus to grow;
(AAV) Nondividing cells location difcult to remove helper virus
Herpes simplex virus 3040 kb 1 x 10
9
Dividing cells Transient No gene expression
Nondividing cells during latent infection
Vaccinia virus 25 kb 3 x 10
9
Dividing cells Transient Potential cytopathic effects
Viral Attributes
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Virus-Based Methods
Viral Workow
Determine viral titer (1 week)
Collect virus
4872 hr
810 hr
Select cells and analyze
Infect with diluted virus
Plate target cells
Target Cell Infection
18 hr
23 days
Transfect
Expression
vector
Virus Preparation
Harvest
supernatant
Add the viral
supernatant to
the target cells
to produce a
stably expressed
cell line
Transfect a 293 producer
cell line with the gene of
interest inserted into the
expression vector
Determine
the titer of
virus stock
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Pros and Cons
Advantages of Virus-Based Methods

Very high gene delivery efciency, 95100%

Simplicity of infection
Disadvantages of Virus-Based Methods

Labor intensive

Best for introducing a single cloned gene that is to be highly expressed

P2 containment required for most viruses
Institutional regulation and review boards required
Viral transfer of regulatory genes or oncogenes is inherently dangerous
and should be carefully monitored
Host range specicity may not be adequate

Many viruses are lytic

Need for packaging cell lines
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Transfection
Workow
Terminology
Life Science
Group
10-0826 0111 Sig 1110 Bulletin 6057 Rev A US/EG
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100
India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000 Vietnam 84 8 38131140
Transfection Methods
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