The Selection Of Transgenic Bovine Fetal Fibroblasts With An Insertional Targeting Vector

The application of sex-sorted semen permits the dairy industry to produce more proportion of femal cattle to take advantage of the lactation capacity. Currently, the high cost, low production rates and low fertility prevent the sperm sexing technology from large scale commercial application.The object of this study was to insert the GFP gene into the Y chromosome of Hostein bulls to produce GFP-expressing Y chromosom bearing sperm by Homologous recombination, which would improve the sperm sexing rate and make less damage to the soted sperm.In this study, bovine fetus fibroblasts was isolated from one male Hostein fetus at 40d pregnancy, the sex of which was identified by PCR. The 1952bp fragment without any loxp site at the 5'end was amplified from the vector p079 pPNT6. After double digestion and ligation, the amplified fragment was used to replace the corresponding fragment on the vector p079 pPNT6, and the backbone vector pPNT-loxp was constructed. The 4147bp fragment with one loxp site at the 3'end and the 3152bp fragment with one lox2272 site at the 5'end were amplified form the genomic DNA of the male fetus. After double digestion and ligation, the two amplified fragments were inserted into the vector pPNT-loxp and the insertional targeting vector pPNTdloxp/USP9Y was constructed.With similar method above, one loxp site and one lox2272 site were inserted into the vector pEGFP-N3 and the vector pEGFP-loxp-lox2272 was constructed. With the mediation of Cre Recombinase, the CMV-EGFP fragmenr on the the vector pEGFP-loxp-lox2272 can be exchanged into the Y chromosome of the gene targeting fibroblasts.The bovine fetus fibroblasts were transfected with BamHI Ilinearized pPNTdloxp/USP9Y by LipofectamineTM 2000. 24 hour later, all the cells were receeded in 96-well plates or 48 -well plates and selected with cell culture medium with 600μg / mL G418. After selection and expansion, eighty-two G418 resistant cell colonies were obtained, one of them were gene targeted cell clone after PCR identification.