Estimating the copy numbers of exogenous gene in transgenic cashmere goats by real-time fluorescence quantitative PCR / 中国兽医学报

ABSTRACT

The copy numbers of exogenous gene in transgenic animals is always regarded as an important information of transgenic animals.Thus,simple and sensitive methods are required for the detection of the copy numbers of exogenous gene.Three kinds of transgenic Shanbei white cashmere goats,containing Tβ4-GFP,FGF5s-GFP and VEGF164-GFP,has been obtained by using PiggyBac(PB) transposon system.Fluorescence quantitative PCR was carried out to detect the copy numbers of copGFP.Using Gluc as reference gene,the double standard curves of exogenous gene and reference gene were mapped and the genomic DNA of transgenic goats were analysized by real-time fluorescence quantitative PCR.Moreover,the copGFP/Gluc ratio in the samples was calculated as the copy numbers of copGFP.In addition,Tβ4-GFP transgenic cashmere goats were selected to detect the integration sites by using the genomic walking kit.The results showed that the standard curve equation of copGFP was y=-3.230 6x+39.216 (R2 =0.998 8) and the standard curve equation of Gluc was y=-3.564 8x+38.440 (R2 =0.996 0).The copy numbers of exogenous gene in the transgenic cashmere goats were obtained and the numbers of integration sites in the selected Tβ4-GFP transgenic goats were consistent with the copy numbers of copGFP.As a conclusion,the high throughput,fast and sensitive real-time fluorescence quantitative PCR is an efficient and convenient method for the copy number of exogenous gene in transgenic cashmere goats.