Transfection Methods Overview Transfection Methods
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Transfection Methods Overview Transfection Methods
Overview
1 Terminology 4 Common Transfection Methods Reagent-Based Methods Lipids 2 Factors Affecting Transfection Calcium phosphate Host Cell Cationic polymers Cell health DEAE-dextran Cell culture Activated dendrimers
Genetic Material Magnetic beads DNA quality and quantity Instrument-Based Methods Electroporation Biolistic technology Microinjection Laserfection/optoinjection
Virus-Based Methods
Overview 2
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Terminology
Terminology 3
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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 (24–96 hours).
Terminology 4
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Host Cell Cell health Cell culture
Genetic Material 2 DNA quality and quantity
Factors Affecting Transfection
Factors Affecting Transfection 5
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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 37°C with CO2 supplied at the correct percentage (5–10%) and 100% relative humidity
■■ Cells should be maintained in log phase growth
Factors Affecting Transfection 6
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Cell Culture
Confluency and Growth Phase
■■ Cells should be transfected at 40–80% confluency (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
Factors Affecting Transfection 7
www.bio-rad.com/transfection DNA Quality Transfection Methods Genetic Material and Quantity
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 final concentration of 0.2–1 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
Factors Affecting Transfection 8
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Transfection Workflow
Transfection Workflow 9
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Transfection Workflow
Tissue culture Count cells
Resuspend cells in electroporation buffer
Add nucleic acid
Transfect cells
Plate cells
Protein expression Analysis Gene expression
Western blot analysis Reporter gene activity Microscopy Flow cytometry Real-time qPCR
Transfection Workflow 10
www.bio-rad.com/transfection TransfectionTransfection Methods Methods | Overview
Reagent-Based Methods Lipids Calcium phosphate Cationic polymers DEAE-dextran Activated dendrimers Magnetic beads 4 Instrument-Based Methods Electroporation Biolistic technology Common Microinjection Transfection Laserfection/optoinjection Methods Virus-Based Methods
Common Transfection 11 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Lipids
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
Common Transfection 12 Methods
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Lipid-Mediated Nucleic acid
Gene Delivery Cationic lipid Transfection Complexing Lipid-mediated gene delivery is also referred to as lipofection, or liposome-based gene transfection. It uses lipids to cause a cell Liposome to absorb exogenous DNA. Endocytosis Transfer of genetic material into the cell takes place via
liposomes, which are vesicles that can merge with the cell Analysis membrane since they are both made of a phospholipid bilayer.
Common Transfection 13 Methods
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Method Overview
Solution A Solution B
2–4 µl Transfectin™ lipid reagent 0.25–1 µg plasmid DNA per 50 µl serum-free medium per 50 µl 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 µl/24-well plate)
Plated cells Aspirate medium from cells Incubate overnight and assay
Common Transfection 14 Methods
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Pros and Cons
Advantages of Lipids
■■ Deliver nucleic acids to cells in a culture dish with high efficiency
■■ 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
Common Transfection 15 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Calcium Phosphate
Calcium Phosphate Ca++
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 Ca++ at room temperature. Ca++
This step generates a precipitate that is dispersed onto the Ca++ Ca++ cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. Calcium phosphate
Ca++
DNA
Common Transfection 16 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Calcium Phosphate
Method Overview
Solution A: DNA in calcium solution Solution A Solution B Solution B: 2x Hanks buffered saline solution
1 Add solution A to solution B while vortexing.
Step 1 2 Incubate 20–30 min. Apply the solution to a subconfluent cell culture.
Step 2 3 Incubate 2–12 hr. Replace the solution with complete growth medium.
Step 3 4 Assay for transient gene expression or begin selection for stable transformation time.
Step 4
Common Transfection 17 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Calcium Phosphate
Pros and Cons
Advantages of Calcium Phosphate
■■ Inexpensive
■■ High efficiency (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 efficiency
■■ 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
Common Transfection 18 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Cationic Polymers
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 efficiently 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.
Common Transfection 19 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods DEAE-Dextran
DEAE-DextranHeadline
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.
CH2 CH3 + Dextran O CH2 CH2 N H
CH2 CH3 Diethylaminoethyl
Common Transfection 20 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods DEAE-Dextran
Solution A Solution B MethodHeadline Overview Step 1
Solution A: DNA (~1–5 µg/ml) diluted into 2 ml of growth Step 2 medium with serum containing chloroquine
Solution B: DEAE-dextran solution (~50–500 µg/ml) Solution C: ~5 ml of DMSO Step 3 Solution D: Complete growth medium Solution C
Step 4 1 Add solution A to solution B, then mix gently.
2 Aspirate cell medium and apply the mixed A and B solutions to the subconfluent cell culture. Incubate the DNA mixture for ~4 hr.
Solution D 3 Aspirate supernatant.
Step 5
4 Add solution C to induce DNA uptake.
5 Remove DMSO and replace with complete growth medium; assay for transient gene expression.
Common Transfection 21 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods DEAE-Dextran
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 efficiencies will vary with cell type
■■ Can be used only for transient transfection
■■ Typically produces less than 10% delivery in primary cells
Common Transfection 22 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Activated Dendrimers
G4 Generation Activated numbers
Dendrimers Structure G3 G2 Positively charged amino groups (termini) on the surface of Core G1 the dendrimer molecule interact with the negatively charged G0 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 Branching Termini membrane of eukaryotic cells. Complexes bound to the cell points surface are taken into the cell by nonspecific endocytosis. Once inside the cell, the complexes are transported to the endosomes. Dendrimer 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.
Common Transfection 23 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Magnetic Beads
Magnet-Mediated Transfection
Magnet-mediated transfection uses magnetic force to deliver DNA into target cells. Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid–particle complexes toward and into the target cells, where the cargo is released.
Common Transfection 24 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Magnetic Beads
Negatively HeadlineMethod Overview charged DNA
1 Dilute nucleic acid in medium.
Positively charged 2 Add magnetic nanoparticle. magnetic particles
3 Incubate 10–20 min.
4 Add medium to adherent cells (2–4 x 105 cells).
5 Add nucleic acid/nanoparticle solution.
6 Place culture plate on magnet plate.
7 Incubate 15 min.
8 Remove magnet plate.
Common Transfection 25 Methods
www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Magnetic Beads
Pros and Cons
Advantages of Magnetic Beads
■■ Rapid
■■ Increased transfection efficiency 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
Common Transfection 26 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Electroporation
Step 1 + Ions Gene – Cell How Electroporation Works membrane
1 Electroporation exposes a cell to a high-intensity electric field that temporarily Before Field Applied destabilizes the membrane. Step 2 + –
2 During this time the membrane is highly permeable to exogenous molecules present in the surrounding media.
Field Applied — Ions Move 3 DNA then moves into the cell through these holes. Step 3 + –
4 When the field is turned off, the pores in the membrane reseal, enclosing the DNA inside.
Field Applied — Pathways Form Step 4 + –
Field Removed — Membrane Heals
Common Transfection 27 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Electroporation
Type of Electrical Pulse — the Waveform
The most common electrical fields are exponential and square waveforms. The waveform has a significant effect on the transfection efficiency for different cell types. Both exponential-decay and square-wave pulses have been used very effectively for electroporation.
V0 V0 V0 Vt V0 Vt Voltage, V Voltage, Voltage, V Voltage, Voltage, V Voltage, t V Voltage, t
V0 V0 e e
t t Time, ms Time, ms Time, ms Time, ms
Exponential Waveform Square Waveform
■■ Capacitors charged to a set voltage ■■ Determined by pulse duration and/or number of pulses
■■ Set voltage is released from the selected capacitor ■■ Several short pulses may be more beneficial than one long pulse and decays rapidly (exponentially) over time
Common Transfection 28 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Electroporation
Pros and Cons
Advantages of Electroporation
■■ Nonchemical method that doesn’t seem to alter the biological structure or function of the target cells
■■ Easy to perform
■■ High efficiency
■■ Can be applied to a wide range of cell types
Disadvantage of Electroporation
■■ Cell mortality (if using suboptimal conditions)
Common Transfection 29 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Biolistic Technology
Biolistic Particle Delivery
Biolistic transformation is the delivery of nucleic acids into cells via high velocity nucleic acid–coated microparticles.
Helios® Gene Gun PDS-1000/He™ Biolistic Particle Delivery System
■■ For in situ, in vivo, and in vitro transformations ■■ For in vitro, ex vivo, and in vivo (for some plants and microbes)
■■ Applications for animals, plants, cell culture, nematodes, transformations
yeast, and bacteria ■■ Applications for animal cell and organ cultures, plant cell cultures
■■ Pressure range 100–600 psi enables fine-tuning of penetration and explants, pollen, insects, algae, fungi, and bacteria
■■ Highly portable — can be used in the field ■■ Pressure range 450–2,200 psi gives flexibility and penetration —
■■ Small target area for accurate targeting ideal for plant applications
■■ Large target area — more cells can be transformed
Common Transfection 30 Methods
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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.
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6
Common Transfection 31 Methods
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PDS-1000/He™ System — Process Overview
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.
Gas acceleration tube
Rupture disk Macrocarrier Stopping screen Microcarrier launch assembly
DNA-coated microcarriers
Target cells Target shelf
Common Transfection 32 Methods
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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 cm2) Large (40 cm2) Largest (~75 cm2)
Pressure range 100–600 psi 450–2,200, psi 450–2,200 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, Plants: Small intact plants, Plants: Cells with thin cell walls explant, and organ culture plant cell culture, explants Yeast, bacteria, other microbes Plants: Field and greenhouse Yeast, bacteria, other microbes use, plant cell culture, explants Organelles (chloroplasts, Yeast, bacteria, other microbes mitochondria, etc.)
Common Transfection 33 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Biolistic Technology
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 efficiency compared to electroporation or viral- or lipid-mediated transfection
■■ Limited bacterial transfection data
■■ Preparation of microparticles
■■ Instrument cost
■■ Requires purchase agreement
Common Transfection 34 Methods
www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Microinjection
Microinjection
■■ Direct injection of naked DNA
■■ Laborious (one cell at a time)
■■ Technically demanding and costly
■■ Can be used for many animals
Holding pipet Injecting pipet
Transgene
Fertilized egg, 100 µm
Common Transfection 35 Methods
www.bio-rad.com/transfection Laserfection/ Transfection Methods Instrument-Based Methods 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 efficiently optoinjected, including ions, small molecules, dextrans, short interfering RNAs (siRNAs), plasmids, proteins, and semiconductor nanocrystals, into numerous cell types
■■ Advantages: very efficient; works with many cell types; fewer cell manipulations needed
■■ Disadvantages: requires the cells to be attached; expensive laser-based equipment needed
Impermeant nanoparticles
Intact cell 532 nm laser beam Transiently permeabilized Intact cell
Common Transfection 36 Methods
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Viral Vectors
Retroviruses
■■ Murine leukemia virus (MuLV)
■■ Human immunodeficiency 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 specific 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.
Common Transfection 37 Methods
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Viral Attributes
Viral Vector DNA Insert Size Maximum Titer Cell Type Expression Pitfalls
Retroviral 8 kb 1 x 109 Dividing cells Stable Random insertion site
Lentivirus 9 kb 1 x 109 Dividing cells Stable Random insertion site Nondividing cells
Adenovirus 8 kb 1 x 1013 Dividing cells Transient Highly immunogenic Nondividing cells
Adeno-associated virus 5 kb 1 x 1011 Dividing cells Stable, site-specific Requires helper virus to grow; (AAV) Nondividing cells location difficult to remove helper virus
Herpes simplex virus 30–40 kb 1 x 109 Dividing cells Transient No gene expression Nondividing cells during latent infection
Vaccinia virus 25 kb 3 x 109 Dividing cells Transient Potential cytopathic effects
Common Transfection 38 Methods
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Virus Preparation Viral Workflow Transfect a 293 producer cell line with the gene of interest inserted into the Transfect expression vector 8–10 hr
Expression vector
Target Cell Infection
Plate target cells
48–72 hr
Harvest Collect virus supernatant Add the viral supernatant to 18 hr the target cells to produce a stably expressed Infect with diluted virus cell line
Determine Determine viral titer (1 week) the titer of virus stock 2–3 days
Select cells and analyze
Common Transfection 39 Methods
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Pros and Cons
Advantages of Virus-Based Methods
■■ Very high gene delivery efficiency, 95–100%
■■ 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 specificity may not be adequate
■■ Many viruses are lytic
■■ Need for packaging cell lines
Common Transfection 40 Methods
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