The Establishment Of Immortalization Of Dairy Goat Mammary Epithelial Cells

Epthelial cells is a highly differentiated somatic cells, it is difficult to culture GMECs forlong time in vitro, But it still maintain the function of milk synthesis and secretion in vitroculture conditions, so it can be used as target cells of the mammary gland bioreactor and it canproduce exogenous protein product which have a high medicinal value. It is very meanningfμlto obtain transgenic animals by nuclear transfer and establishment of mammary glandbioreactor. Therefore, the establishment of a potential immortalized mammary epithelial cellline is very important. There are many immortalization methods, such as transfection of SV40large antigen, HPV E6and E7, silence of anti-ongene, overexpression of ongene and so on.But using human telomerase reverse transcriptase gene can effectively reduce the risk ofchanges in the biological characteristics of cells in the process of immortalization or evencancer. The aim of the present study was to immortalize BMECs by hTERT. We got theresults as followed:⑴Bovine mammary epithelial cells had been successfully separated in the test, and thecells could be stained by Keratin. In vitro, the cells could secret β-casein under inductioncondition.⑵Use liposomal transfection method to transfect pCI-neo-hTERT into thethird-generation dairy goat mammary epithelial cells. Now it spread to52generations.Through Immunofluorescence assay and RT-PCR,we detected the expression of hTERT,indicating that the foreign gene has been successfully integrated into the mammary epithelialcells and have stable expression.⑶Induce the50th generations of hTERT-GMECs by adding hormone stimulation,through RT-PCR to detect the expression of β-casein, the results indicate that hTERT-GMECscan still express β-casein, indicating that cell secretory function normal.