NDLI: Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts

Content Provider	Springer Nature Link
Author	Ross, Jason W. Whyte, Jeffrey J. Zhao, Jianguo Samuel, Melissa Wells, Kevin D. Prather, Randall S.
Copyright Year	2009
Abstract	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 μL of 1 × 10$^{6}$ cells/mL in the presence of 12.5 μg 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.
Starting Page	611
Ending Page	620
Page Count	10
File Format	PDF
ISSN	09628819
Journal	Transgenic Research
Volume Number	19
Issue Number	4
e-ISSN	15739368
Language	English
Publisher	Springer Netherlands
Publisher Date	2009-11-24
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Transgenic swine Electroporation efficiency Transfection efficiency eGFP Human Genetics Plant Sciences Animal Genetics and Genomics Plant Genetics & Genomics Molecular Medicine
Content Type	Text
Resource Type	Article
Subject	Genetics Animal Science and Zoology Biotechnology Agronomy and Crop Science

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