Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts.
Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 +/- 0.8% to 43.0 +/- 3.4% while survival decreased from 90.5 +/- 8.0% to 44.8 +/- 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1-5 pulses from 63.2 +/- 6.3% to 3.0 +/- 0.3% with GFP expression of surviving cells increasing from 35.6 +/- 2.67% to 71.4 +/- 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 muL of 1 x 10(6) cells/mL in the presence of 12.5 mug DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.
Ross
Jason W
JW
Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
Whyte
Jeffrey J
JJ
Zhao
Jianguo
J
Samuel
Melissa
M
Wells
Kevin D
KD
Prather
Randall S
RS
D000818:Animals; D030801:Animals, Genetically Modified; D002138:Calibration; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary; D004351:Drug Resistance / Q000235:genetics; D018274:Electroporation / Q000379:methods* / Q000592:standards; D004622:Embryo, Mammalian; D005333:Fetus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D053652:Nuclear Transfer Techniques / Q000592:standards / Q000662:veterinary; D013552:Swine / Q000196:embryology; D014162:Transfection / Q000379:methods / Q000592:standards; D019076:Transgenes
D049452:Green Fluorescent Proteins; D009355:Neomycin