Research on gene targeting to multiple loci in cow fetal fibroblasts

The repetitive ITS(internal transcribed spacers) between rDNA genes were used as the target loci for gene targeting to multiple loci in cow fetal fibroblasts.The techniques of gene targeting to multiple loci using rDNA repetitive sequences as target loci have been set up.The nuclei donor cells for cloning the transgenic cows have been obtained.Firstly,the cow fetal fibroblasts have been isolated,cultured,identified the sex and analysed the karyotypes.The lowest efficient concentrations of G418 and GCV for the positive-negtive selection have been affirmed by MTT method.Secondly,the 7 cell clones expressing green fluorescence have been got by transfect of the gene targeting vectors to multiple loci and positive-negtive selection.One of the 7 cell clones was confirmed as cell clone of gene targeting in site and GFP expression by PCR,RT-PCR and DNA sequencing.