Cre/Loxp Mediated Deletion Of Maker Gene In Transgenic Cashmere Goat

The development of nuclear transfer and transgenic technology has provide a new way for the cultivation of transgenic animal varieties. The target gene and marker gene were transfected into the cell in order to select and identify transgenic cell efficiently. Red fluorescent genes and resistance gene were commonly used. The application of marker gene has attracted attention and concern about its security. The purpose of this study is to delete the maker gene by using the Cre/loxp recombination system.In this study, VEGF164gene was linked with promoter K14, both ends with same direction loxp side. When this fragment was linked with vector pDsReD2-1, the expression vector pDsReD2-1-K14-VEGF-LOXP was constructed successfully. After the expression vector was transferred into goat fetal fibroblasts, high purity of transgenic cell clones were selected. Part of the cell clones were used as donor cells for nuclear transfer to produce transgenic cloning embryos. After embryo transfer, the transgenic goat was delivered, and its somatic cells can be used in the following research to delete marker gene. The other part of the cell clones were directly treated with recombinant Cre (HTNC), which linked with signal peptide fragments, then the removal efficiency was detected. Finally the cells treated under different time were collected, real time fluorescence quantitative PCR was used to evaluate the removal efficiency of marker gene. The result showed that the marker gene was removed partly, and the optimum treatment time was3hours.By the method of this study, it not only can produce a large number of transgenic donor cells for nuclear transfer, but also has important significances in further research to cultivate transgenic goat which comply with GMO biosafety.