Targeting efficiency of α-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Dong Il Jin, Seung Hyeon Lee, Jin Hee Choi, Jae Seon Lee, Jong Eun Lee, Kwang Wook Park, Jeong Sun Seo

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


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 α-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neor) 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.

Original languageEnglish
Pages (from-to)572-577
Number of pages6
JournalExperimental and Molecular Medicine
Issue number6
StatePublished - 31 Dec 2003


  • Animal cloning
  • Galactosyltransferases
  • Gene targeting
  • Swine


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