Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells.
Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.
Jin
Dong-Il
DI
Ilchun Molecular Medicine Institute, Medical Research Center, Seoul National University, Korea.
Lee
Seung-Hyeon
SH
Choi
Jin-Hee
JH
Lee
Jae-Seon
JS
Lee
Jong-Eun
JE
Park
Kwang-Wook
KW
Seo
Jeong-Sun
JS
D000818:Animals; D005347:Fibroblasts; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D016133:Polymerase Chain Reaction; D013552:Swine / Q000196:embryology* / Q000235:genetics
D005700:Galactosyltransferases