Delivery and integration of DNA

Table 20.2 summarizes the major methods for delivery of homologous recombination vectors. Electroporation is the standard method for transfecting mESCs and has been used successfully to produce thousands of mESC clones that remain pluripotent following electroporation, drug selection, screening, and expansion. An alternative to electroporation, preferred by some labs, is lipid-mediated DNA transfection, also called lipofection. Lipofection is a passive method for the introduction of DNA into cells, using synthetic cationic lipids that interact with DNA to form lipid/DNA complexes,

Table 20.2 Methods used to genetically modify hESCs Date cell line Observation

Reference

Transgenesis: random integration, gain of function

Lipofection: ExGen500

(1) Murine Rex1 promoter-eGFP

(2) Murine PGK-eGFP

SV40 driving neo resistance (200 |ig/mL G-418) 13 days of selection. Different methods of transfection were tested; lipofectamine, electroporation, FuGene, ExGen (ExGen 500 was found to be most efficient in this study) Transgenesis: random integration, gain of function HSV-tk via pPNT vector Lipofection: ExGen500

Expression of HSV-tk renders the cells sensitive to ganciclovir and provides a mechanism to selectively eliminate the population expressing this gene. Tumors in mice could be eliminated by treatment with ganciclovir. Culture: KD-MEM, 20% KSR, 4ng/mL FGF-2

107 cells, 12 |ig of plasmid, selected with G418, 10 days after selection Results: 9 colonies, 6 of which were sensitive to ganciclovir Homologous recombination: loss of function

Electroporation

(1) Knockout: HPRT1

12 kb of homology gave 22 hprt-negative clones out of 56 stable clones

Homologous recombination: loss of function

Lipofection: ExGen 500 Knockout replacement vector: HPRT1

Human ESCs selected hygromycin (100 |ig/mL) and 6TG (1 |ig/mL) containing medium, clones isolated after 3 weeks

Table 20.2 (Continued) Date cell line Observation

2004 WA09

2004

WA01

WA09

2005 HES3

2006 WA09.2

Reference

Transgenesis: gain- and loss-of-function study: GFP, RFP, and siRNA

Lipofection

Lipofectamine 2000 and ExGen 500 (found Lipofectamine 2000 more effective)

Produced transient siRNA and stable hairpin-loop siRNA

Evaluated five different red and green fluorescent proteins for stable long-term expression and ability to knock down expression with siRNAs

Transgenesis: random integration: gain of function

Lipofection

FuGene 6

10 |ig linearized plasmid, 24 h after transfection, selected with 100 |ig/mL G418, after 12 days a single eGFP-expressing colony was observed. It was further propagated in MEF-CM containing G418 for four months. Plasmids: EF1a driving eGPF and EF1a driving neomycin resistance Transgenesis: random integration

Electroporation

"Envy"

hpactin-GFP

Mapped to chromosome 12 q23.1 between the genes encoding thrombopoietin (TPO) and solute carrier 25 (SC25)

Normal, pluripotent, and uniformly expressed GFP in derivatives of all three germ layers in the absence of ongoing selection

Transgenesis: two-step generation of Cre-modified human ESC clones

Electroporation to generate stable transgenic clones: 40 |ig plasmid; 1-3 X 107 cells; 4 mm gap cuvette; 500 |iL culture medium + 300 |iL PBS; 300 V, 200 |iF, 5-20 ms pulse time; 300 |ig/mL G418 selection started 48 h after electroporation Lipofection for transient transfection of Cre plasmid

Vallier et al., 2004

Costa et al., 2005

Nolden et al., 2006

which fuse with the cell membrane and deliver the DNA to the cell. Lipofection can be used for transient expression as well as stable expression. Both methods have been used on hESCs, and sample protocols are outlined in the following section. Viral infection techniques have been used extensively for hESC transformation, but the relatively small capacity of these vectors makes it difficult to use them for targeting strategies that require large regions of homologous sequence (see Chapter 19).

Electroporation

Differences between electroporation in mouse and human ESCs have not been carefully examined, but a well-known problem with hESCs is that, unlike mESCs, the cells do not survive well in single-cell suspension. Electroporation causes massive cell death, so it is difficult to separate the effects of the electroporation itself from the effects of cell dissociation. This makes systematic testing of electroporation protocols difficult, and in the techniques that have been used so far, cells are electroporated in multicell clumps rather than as single cells.

Lipofection

Lipid-mediated methods do not require dissociation to single cells, but the cells must be accessible to the lipid vesicles, so this method does not work well for multilayered cultures. Experiments with mESCs show that some dissociation of the cells is required for optimal transfection efficiency, and transfection may work better in suspension cultures than in attached cells. Several manufacturers have lipofection products, and each should be tested on the cells that are to be transfected, varying DNA/lipid ratios, concentrations, and incubation times. Commercially available lipid mixtures include FuGene 6 (Roche Diagnostics), ExGen 500 (Fermentas), and Lipofection 2000 (Invitrogen).

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  • Prisca Longhole
    What is the difference between electroporation and lipofection?
    8 years ago

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