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www.bio-rad.com/transfection Transfection Methods Overview Transfection Methods Overview 1 4 Terminology 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 3 Biolistic technology Transfection Workflow Microinjection Laserfection/optoinjection Virus-Based Methods Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 2 www.bio-rad.com/transfection Transfection Methods 1 Terminology Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 3 www.bio-rad.com/transfection Transfection Methods Terminology 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). Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 4 www.bio-rad.com/transfection Transfection Methods Host Cell Cell health Cell culture Genetic Material 2 DNA quality and quantity Factors Affecting Transfection Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 5 www.bio-rad.com/transfection Transfection Methods Host Cell Cell Health 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 ■■ ■■ Home ells should be incubated at 37°C with CO2 supplied at the correct percentage C (5–10%) and 100% relative humidity Cells should be maintained in log phase growth Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 6 www.bio-rad.com/transfection Transfection Methods Host Cell Cell Culture 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 ■■ ctively dividing cells take up DNA better than quiescent cells (breakdown and perforation A of the nuclear membrane during mitosis enable nuclear delivery) Number of Passages for Primary Cells The number of passages should be low (<50) ■■ ■■ ■■ ■■ Home The number of passages for cells used in a variety of experiments should be consistent ell characteristics can change over time with immortalized cell lines and cells may not C respond to the same transfection conditions Cells may not respond to the same transfection conditions after repeated passages Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 7 www.bio-rad.com/transfection Transfection Methods DNA Quality and Quantity Genetic Material DNA Quality and Quantity ■■ ■■ ■■ Home 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 he optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA, T transfection reagent/method, target cell line, and number of cells Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 8 www.bio-rad.com/transfection Transfection Methods | Overview Transfection Methods 3 Transfection Workflow Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 9 www.bio-rad.com/transfection Transfection Methods Transfection Workflow Transfection Workflow Tissue culture Count cells Resuspend cells in electroporation buffer Add nucleic acid Transfect cells Plate cells Protein expression Western blot analysis Home Overview Analysis Reporter gene activity Terminology Factors Affecting Transfection Microscopy Transfection Workflow Gene expression Flow cytometry Common Transfection Methods Real-time qPCR 10 www.bio-rad.com/transfection Transfection Methods | Overview Transfection Methods Reagent-Based Methods Lipids Calcium phosphate Cationic polymers DEAE-dextran 4 Activated dendrimers Magnetic beads Instrument-Based Methods Electroporation Biolistic technology Common Transfection Methods Home Overview Terminology Microinjection Laserfection/optoinjection Virus-Based Methods Factors Affecting Transfection Transfection Workflow Common Transfection Methods 11 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 ■■ Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 12 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Lipid-Mediated Gene Delivery Nucleic acid 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 to absorb exogenous DNA. Liposome Endocytosis 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. Home Overview Terminology Factors Affecting Transfection Lipids Transfection Workflow Analysis Common Transfection Methods 13 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Lipids Method Overview Solution A Solution B 2–4 µl Transfectin™ lipid reagent per 50 µl serum-free medium 0.25–1 µg plasmid DNA 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 Home Overview Aspirate medium from cells Terminology Factors Affecting Transfection Transfection Workflow Incubate overnight and assay Common Transfection Methods 14 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Lipids 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 ■■ Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 15 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 at room temperature. Ca++ Ca++ This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. Ca++ Ca++ Calcium phosphate Ca++ DNA Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 16 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 Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 17 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 ■■ Home alcium phosphate precipitation does not work in RPMI, due to the high C concentration of phosphate within the medium Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 18 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. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 19 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods DEAE-Dextran Headline 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. CH2 CH3 Dextran O CH2 CH2 + N H CH2 CH3 Diethylaminoethyl Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 20 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods DEAE-Dextran Solution A HeadlineOverview Method Step 1 Solution A:DNA (~1–5 µg/ml) diluted into 2 ml of growth medium with serum containing chloroquine Solution B: Solution B Step 2 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 R emove DMSO and replace with complete growth medium; assay for transient gene expression. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 21 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 ■■ Home ■■ Transfection efficiencies will vary with cell type ■■ Can be used only for transient transfection ■■ Typically produces less than 10% delivery in primary cells Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 22 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Activated Dendrimers Structure Activated Dendrimers G4 Generation numbers G3 G2 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 DNAdendrimer 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 nonspecific endocytosis. Once inside the cell, the complexes are transported to the endosomes. Core G1 G0 Branching points Termini 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. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 23 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. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 24 www.bio-rad.com/transfection Transfection Methods Reagent-Based Methods Magnetic Beads Negatively charged DNA HeadlineOverview Method 1 Dilute nucleic acid in medium. Positively charged magnetic particles 2 Add magnetic nanoparticle. 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. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 25 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 igh transfection rates for adherent mammalian cell lines and primary cell cultures H (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 ■■ ■■ Home Requires adherent cells; suspension cells need to be immobilized or centrifuged Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 26 www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Step 1 + How Electroporation Works Ions Electroporation Gene – Cell membrane Before Field Applied 1Electroporation exposes a cell to a high-intensity electric field that temporarily destabilizes the membrane. Step 2 + – 2During this time the membrane is highly permeable to exogenous molecules present in the surrounding media. Field Applied — Ions Move 3DNA then moves into the cell through these holes. Step 3 + – 4When 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 Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 27 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 e Vt Voltage, V Voltage, V Voltage, V V0 t V0 e t t Time, ms ■■ Terminology Time, ms Time, ms t Time, ms ■■ ■■ Overview Vt Square Waveform Determined by pulse duration and/or number of pulses Several short pulses may be more beneficial than one long pulse Exponential Waveform Capacitors charged to a set voltage Set voltage is released from the selected capacitor and decays rapidly (exponentially) over time Home V0 Voltage, V V0 ■■ Factors Affecting Transfection Transfection Workflow Common Transfection Methods 28 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) ■■ Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 29 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 For in situ, in vivo, and in vitro transformations Applications for animals, plants, cell culture, nematodes, yeast, and bacteria Pressure range 100–600 psi enables fine-tuning of penetration Highly portable — can be used in the field 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 450–2,200 psi gives flexibility and penetration — ideal for plant applications Large target area — more cells can be transformed ■■ ■■ ■■ ■■ Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 30 www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Biolistic Technology 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 Home Step 2 Overview Step 3 Terminology Factors Affecting Transfection Step 4 Transfection Workflow Step 5 Common Transfection Methods Step 6 31 www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Biolistic Technology 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. 3The 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 Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 32 www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Biolistic Technology 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 to barrel (skin, organs); cell, Plants: Small intact plants, explant, and organ culture plant cell culture, explants Plants: Field and greenhouse use, plant cell culture, explants Yeast, bacteria, other microbes Home Overview Terminology Factors Affecting Transfection Animals: Cell and organ culture Plants: Cells with thin cell walls Yeast, bacteria, other microbes Yeast, bacteria, other microbes Organelles (chloroplasts, mitochondria, etc.) Transfection Workflow Common Transfection Methods 33 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 ■■ ■■ ■■ ■■ ■■ Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 34 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 Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 35 www.bio-rad.com/transfection Transfection Methods Instrument-Based Methods Laserfection/ Optoinjection Laserfection/Optoinjection ■■ ■■ This procedure uses laser light to transiently permeabilize a large number of cells in a very short time arious substances can be efficiently optoinjected, including ions, small molecules, dextrans, short interfering V 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 Home Overview 532 nm laser beam Terminology Factors Affecting Transfection Transiently permeabilized Transfection Workflow Common Transfection Methods Intact cell 36 www.bio-rad.com/transfection Transfection Methods Virus-Based Methods 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. Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 37 www.bio-rad.com/transfection Transfection Methods Virus-Based Methods 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 Nondividing cells Stable Random insertion site Adenovirus 8 kb 1 x 1013 Dividing cells Nondividing cells Transient Highly immunogenic Adeno-associated virus 5 kb 1 x 1011 (AAV) Dividing cells Nondividing cells Stable, site-specific location Requires helper virus to grow; difficult to remove helper virus Herpes simplex virus 30–40 kb 1 x 109 Dividing cells Transient Nondividing cells No gene expression during latent infection Vaccinia virus Dividing cells Potential cytopathic effects Home 3 x 109 25 kb Overview Terminology Factors Affecting Transfection Transfection Workflow Transient Common Transfection Methods 38 www.bio-rad.com/transfection Transfection Methods Virus-Based Methods Virus Preparation Viral Workflow Transfect a 293 producer cell line with the gene of interest inserted into the expression vector Transfect 8–10 hr Expression vector Target Cell Infection Plate target cells 48–72 hr Harvest supernatant Collect virus 18 hr Infect with diluted virus Determine the titer of virus stock Add the viral supernatant to the target cells to produce a stably expressed cell line Determine viral titer (1 week) 2–3 days Select cells and analyze Home Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 39 www.bio-rad.com/transfection Transfection Methods Virus-Based Methods 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 Home ■■ Many viruses are lytic ■■ Need for packaging cell lines Overview Terminology Factors Affecting Transfection Transfection Workflow Common Transfection Methods 40 www.bio-rad.com/transfection Transfection Methods Bio-Rad Laboratories, Inc. 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