The Effect Of Enhancer On The Expression Of Human LTF In The Goat Mammary Epithelial Cells

This paper studied on whether the specific enhancer sequence and goatβ-lactoglobulin(BLG) regulatory sequences could co-active the expression of human lactoferrin cDNA in the goat mammary epithelial cell by comparing the expression of cell clones which were transfected with two mammary gland-specific expressional vectors independently constructed by different Gene Regulatory Elements.this study offers a reliable guidance to construct highly expressed mammary gland-specific vector and offers the potential- expressed recipient cells to produce highly expressed mammary gland bioreactor in transgenetic animals.Two different mammary gland-specific expressional vector BLC-14 and BnF95 were constructed.The difference between BLC-14 and BnF95 was that BLC-14 has the specific enhancer sequence except for theβ-lactoglobulin(BLG) regulatory sequences.Primary goat mammary cells were obtained using the collegenase I -digesting method . Goat mammary gland tissue from the lactating goat was digested and cultured in medium DMEM/F12 containing 10% fetal calf serum.After a long time observation, it was found that the cell proliferate vigorously in vitro even after 20th passages.BLC-14 and BnF95 gene sequence containing neor gene as the selective marker were transfected into five goat mammary epithelial cell lines in vitro culture by electroporation. After three weeks,neomycin resistant single colonies were isolated and expanded in precence of 400ug/ml G418, 82 colonies with BLC-14 and 73 colonies with BnF95 were obtained respectively, of which were picked out into other 24-well plates to proliferate in 200ug/ml medium. DNA samples from 6 clonies were screened by PCR detection and they had been identified to be transgenic cells. After inducing with luteotropic for 48h, supernate were collected and detected by ELISA. 9 clonies from BLC-14 and 7 clonies from BnF95 both of which could stably express hLTF were obtained,From above,it was indicated that both of the mammary gland-specific expression vector BLC-14, BnF95 could express in the GMEC and they had a significant difference(P<0.05) in expressing levels,which proved that the enhancer -controlled genes could enchance the experssing level of the transgenes in the GMECs...