Study On Expression Of Human Proinsulin Gene Under The Control Of Both Goat BLG Regulatory Region And Enhancer Seaquence

A goatβ-lactoglobulin(BLG) regulatory sequences, enhancer seguence, mark gene (Neo gene) and human proinsulin genomic DNA were used to construct mammary gland expression vector. The construct was transfected into goat mammary epithelial cells(GMECs) by electroporation. After three weeks'selection with G418, we obtained 6 GMECs which can express human proinsulin when the medium was supplemented with lactogenic hormones. It is crucial to produce transgenic goats by somatic cells nuclear transfer in the next study.1.Construction of mammary gland-specific expression vectorFirstly, human proinsulin genomic DNA was cloned from human genome by pyrobest Taq PCR.The size of the gene is 1182bp. The goat BLG regulatory sequences was cloned from goat genome by the long fragment PCR. The size of the gene is 6.9Kb containing the 4.132kb 5'end regulatory region and the 1.805kb 3'end flanking region. The 600bp enhancer sequence was from a commercial plasmid. Neo gene was also from a plasmid of pGH-4. In this study, we finally have constructed a human proinsulin mammary gland-specific expression vector under the control of both goat BLG regulatory sequences and enhancer sequence.2.Culture, transfection and drug selection of GEMCsGMECs were isolated from lactating SaNeng goat mammary glands by the collagenase/hyaluronidase dissociation method. The gene segment from BIC13 was transfected into the 4th passage mammary epithelial cells by electroporation. Then cells were selected with 400μg/ml G418 sulfate for a period of 3 weeks. We obtained 10 cell...