The Construction Of Goat β-lactoglobulin Regulatory Sequence/human Lactoferrin CDNA Fusion Gene, Transfection And Production Of Transgenic Cloned Goat

The research of mammary gland bioreactor is one of the most active areas in biology. The success of somatic nuclear transfer provides a new method for the advancement of mammary gland bioreactor. Compared with the traditional way of microinjection, the superiority of this method is that the transgenic was brought forward to the phase of cell culture, and the somatic cell which was intergrated by vector was used for nuclear transfer. So the born animal would be the transgenic and the efficiency would be greatly improved by this way. Therefore, the present study was undertaken to explore the transfectiom selection and nuclear transfer in goat fetal fibroblasts by using upstream and downstream of goat β -lactoglobulin as regulate gene, human lactoferrin cDNA as integrated gene, Neo gene as mark gene.Firstly, the 4.132kb 5'end regulatory region and the 1.805kb 3' end regulatory region were amplified from goat genome by the long fragment PCR technology. These two fragments were cloned into the pGEM-T easy vector for the sequencing analysis. Homologous sequence alignmentindicated a homology of 99% with the goat sequence which has been published. According to these two fragments, we constructed the mammary specific expressing vector, BFK40.The result of PCR and restriction enzyme digestion indicated that the anticipated construction was correct.Three goat fetal fibroblast lines were established by trypsin digestion. Two of three fetal fibroblast lines were identified to be female and one to be male by the SRY-PCR method. By electroporation, the lineated mammary specific expressing vector, BFK40, were transfected into female fetal fibroblast line. In the end, we succeeded in getting transfected cell lines by selection with G418 and validate with PCR.Lastly, the transfected cell lines were used as donor cells in this study, metaphase Ⅱ (M ⅡI) oocytes were collected from oviducts of superovulated donor goats and enucleated as recipient cytoplasts, reconstructed oocytes were surgically transferred to synchronized surrogate recipients. An offspring was successfully born. PCR-RFLP analysis confirmed that the cloned offspring were derived from the donor cells. The incorporation of BFK40 and the expression of Neo gene were proved by the polymerase chain reaction (PCR) analysis and the selection with G418. All these proved that the offspring was a transgenic cloned goat.