Establishment And Preliminary Application Of Dairy Goat Mammary Gland Epithelial Cell Lines

To establish dairy goat mammary gland epithelial cell lines for evaluation of mammary gland-specific expression gene constructs, a pair of primers were synthesized according to the previously published DNA sequence and used to amplify the SV40 T gene by high fidelity PCR from COS-1 cell genomic DNA. An expected PCR product of 2.5kb was obtained and shown to be 99% identical to that of the previously published SV40 T gene by DNA sequencing. The amplified SV40 T gene was subcloned into eukaryotic expression vector pTarget and the resulting recombinant vector pTarget-LT was used to transfect dairy goat primary mammary gland epithelial cells. Following limited dilution and repeated passage, 18 cell clones were obtained, one of which had been passed for 30 generations in vitro.The cell clone maintained its original morphology of mammary gland epithelial cells and was able to form glandulous structures on collagen matrix with an increased maturation density and growth speed. Dot blotting analyses showed that the SV40 T gene was integrated into the genomes of the transformed cells and that the endogenous β - casein gene was transcribed following induction with a mixture of prolactin, insulin and hydrocortisone. The hormone-dependent expression of the endogenous β - casein gene was confirmed by ELISA using self-made anti goat β - casein immunoserum with a highest concentration of 1 μ g /ml. Further experiments showed that the established cell line was able to support expression of LacZ report gene cloned into mammary gland-specific expression vectors pHC and p205C3. These data indicate that the amplified SV40 T gene maintained its original capability for cell transformation and that the recombinant vector pTarget -LT was usable for establishment of dairy "goat mammary gland epithelial cell lines. The established cell line maintained the original capabilities of differentiation and expression of normal goat mammary gland epithelialcells and was a useful cell mode for evaluation of mammary gland-specific expression gene constructs.