Goat Beta-casein Gene Regulatory Sequences In The Goat Fetal Fibroblast Cells, Homologous Sequences, Gene Targeting Of A Preliminary Study

In this study, with the regulation elements of β -casein gene as homologous sequences, the neo gene as positive selection marker and the HSV-tk gene as negative selection marker, the gene targeting vectors were transfected into the goat fetal fibroblasts to obtain desired targeted fibroblast.The neo gene flanked by direct loxP elements was amplified from the template of pNe vector. After cloned into pGEM-T easy vector, the neo gene was inserted into pBCl vector including the regulative elements of 3 -casein gene and gain recombinant pBN vector. In order to obtain proper length of β -casein regulation sequence homologous fragment for gene targeting, about 4.8kb fragment from pBN vector was cut to gain vector pBCl-S. The HSV-tk gene was cut from vector pGH-4 and spliced to vector pBCl-S to achieve vector pTargetl.The goat primary fetal fibroblasts were isolated from 30-day old fetus, then cultured and passaged into sixteen cell lines. The sex of these cell lines was identified by PCR with amplifying core sequence of goat SRY gene.The linearized pTargetl vectors were transfected into P2 of fibroblasts. The positive cell clones were obtained in both G418 and ganciclovir medium, and called GFFC14-pTargetl. The positive cell clones wereanalyzed by Special-primer PCR. A special band showed in PCR confirmed that there are some gene targeted cells in these positive cell clones.The linearized pBN vectors were introduced into P2 of fibroblasts, and selected with G418.The transfected cells called GFFC14-pBN were used as donor of nuclear transfer to produce transgenic cloned goat. 40 days later after nuclear transfer, three pregnant recipient goats were screened by B-ultrasonic.