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Targeting efficiency of α-1,3-galactosyl transferase gene in pig fetal fibroblast cells
Dong-IlJin,Seung-HyeonLee,Jin-HeeChoi,Jae-SeonLee,Jong-EunLee,Kwang-WookPark,Jeong-SunSeo 생화학분자생물학회 2003 Experimental and molecular medicine Vol.35 No.6
Animal cloning technology with somatic cels pro-vides an alternative tol to conventional methods in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cels for knock-ing-out genes. Homologous recombination events in somatic cells are relatively inefficient as com-pared to those in ES cells, sugesting the need for establishment of efficient gene targeting sys-tem in somatic cels. To investigate the efficiency of positive and negative selection for gene targe-ting in pig fetal fibroblast cells, pig α-1,3-galac-targeting. The neomycin phosphotransferase (Neor) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selec-tion markers in this experiment. Folowing trans-fection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium contain-ing 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 colonies to scren genomic DNA. The frequency of gene targeting in pig fetal fibroblast cels with double drug selection was more than 10-fold efficient compared to that with G418 single sel-ection. Double selection method with Neo and HSV-tk genes could be useful for gene targeting in somatic cels for production of cloned animals carying targeted endogenous genes.