Abstract
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.
Highlights
Recent development of animal cloning technology with somatic cells provides an alternative toolto conventional methodsfor modifyinggenesinanimal (Wilmutetal.,1997).DNA-insertedsomaticcellscan be selected in dishes and nuclear transfer can be done with those cells (Schnieke et al, 1997; Cibelli et al, 1998; Zakhartchenkoet al., 2001)
The double selection procedure developed by Mansouret al. (1988)and Capecchi(1989)hasbeeneffectivelyused forgenetargetingin mouseEScells.Theherpessimplexvirus(HSV)thymidinekinase(tk)geneallowsfor selection againstrandominsertion events withthe selectivedrug,gancyclovirthateliminatesHSV-tkgeneintegratedcellsduringselection.InEScellsystem,the single positive selection results in a 1,000-fold enrichment for targeted cell line and the double selection procedure achieves an additional 10-to 1,000-fold enrichment(Riele,etal.,1992).Thedoubleselection method could have been much more effectivein somatic cells over single selection procedure when strength of cell proliferation is sustained during drug selections
To determine whether positive-negative gene targeting could be achieved in pig fetal fibroblasts,piggenomic13-GTgene wasclonedfrompig genomiclibrary and neomycin phosphotransferase (Neor) plus herpes simplex virus-thymidine kinase (HSV-tk) genes were used for the construction of targeting vector in this experiment.Followingtransfection withtargetingvector DNA,the pigfetalfibroblast cells were selected againstresistance of G418 and gancyclovir
Summary
Recent development of animal cloning technology with somatic cells provides an alternative toolto conventional methodsfor modifyinggenesinanimal (Wilmutetal.,1997).DNA-insertedsomaticcellscan be selected in dishes and nuclear transfer can be done with those cells (Schnieke et al, 1997; Cibelli et al, 1998; Zakhartchenkoet al., 2001). The double (positive/negative) selection procedure developed by Mansouret al. (1988)and Capecchi(1989)hasbeeneffectivelyused forgenetargetingin mouseEScells.Theherpessimplexvirus(HSV)thymidinekinase(tk)geneallowsfor selection againstrandominsertion events withthe selectivedrug,gancyclovirthateliminatesHSV-tkgeneintegratedcellsduringselection.InEScellsystem,the single positive selection results in a 1,000-fold enrichment for targeted cell line and the double selection procedure achieves an additional 10-to 1,000-fold enrichment(Riele,etal.,1992).Thedoubleselection method could have been much more effectivein somatic cells over single selection procedure when strength of cell proliferation is sustained during drug selections. To determine whether positive-negative gene targeting could be achieved in pig fetal fibroblasts,piggenomic13-GTgene wasclonedfrompig genomiclibrary and Neor plus HSV-tk genes were used for the construction of targeting vector in this experiment.Followingtransfection withtargetingvector DNA,the pigfetalfibroblast cells were selected againstresistance of G418 and gancyclovir. Ourresults indicate that the gene targeting in pig fetal fibroblasts can be achieved by using double selection procedure with hightargeting efficiency
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