| Mammary gland bioreactor is a hot issue in biological engineering areas currently. We always hope that the foreign genes could be highly expressed in mammalian cells and individuals when foreign genes are transfected into mammalian cells and the transgenic animals are made. But things go contrary to one's wishes, the foreign genes do not express in many transgenic experiments. Researches show that in many cases the expression of the foreign genes which have been integrated into the genome in mammalian cells are relevant to the transgene silencing. Countries in the world are carrying out extensive research on the mechanism and control of the transgene silencing, in order to overcome it to improve the expression of foreign genes. It has become one of the most important problems to the application of transgenic animals in genetic engineering areas at home and abroad. Therefore, from above, the prevention of transgene silencing has great theoretical and practical significance to enhancing the expression of foreign gene and to improve the success rate of transgenic animals.We constructed a mammary gland-specific expression vector BLC-14, established the goat mammary epithelial cell lines in vitro, transfected the mammary gland-specific expression vector BLC-14 into the goat mammary epithelial cells, obtained transfected cell clonies and treated with TSA. By testing the experssing level of the hLF at cell level, whether the histone deacetylases inhibitor TSA could overcome the transgene silencing and enchance the expression of BLG/hLF gene in the goat mammary epithelial cells was investigated. It provides the basis for the further study of constructing high experssed mammary gland-specific vector and offers the potential foreign genes expression cells to the latter part study of producing high expressed transgenetic animals by somatic cell nuclear transfer.1 Construction of mammary gland-specific expressional vectorcDNA sequence of human lactoferrin gene, 5` and 3` flank regulatory sequence ofβ-lactoglobulin gene, neor gene and the sequence of enhancer which had been harboured in our laboratory were used to constructing mammary gland-specific expressional vector BLC-14.2 Establishment of goat mammary epithelial cell lines in vitroPrimary goat epithelial mammary cells were obtained using collegenase I -digesting method.Goat mammary gland tissue from the middle-lactogening adult goat was digested and cultured in medium DMEM/F12 containing 10% fetal calf serum. By tripsin digesting, the pured mammary epithelial cell line could be obtained after 1～2 passages. After a long time observation, it was found that the cell proliferate vigorously in vitro even after 20th passages.3 Transferring BLC-14 into goat mammary epithelial cell lines and the treatment of TSABLC-14 vectors containing neor gene as the selective marker were transfected into 1th or 2th passage goat mammary epithelial cells by electroporation. After three weeks,neomycin resistant single colonies were formed in 400μg﹒mL-1 G418. 89 colonies were obtained, which were picked out and proliferate in 200μg﹒mL-1 medium. DNA samples from 7 clonies were screened by PCR and all of them had been identified to be the transgenic cells. Then colonies divided into two groups as experimental group and comparative group. After inducing comparative group by 5μM luteotropic and experimental group by 5μM luteotropic and 1μM TSA, BLG/hLF expression was tested by ELISA. The results showed that the ELISA positive of the two group were both 11. The experssing level of the experimental group was 0.781μg﹒mL-1 per 106 cells, and the comparative group was 0.558μg﹒mL-1 per 106 cells.Through statistical analysis, the experssing level of the experimental group which was treated with TSA was significantly higher. From above, it was indicated that histone deacetylases inhibitor TSA could enchance the experssing level of the transgene at cell level. The clonies of the highest expression would be used as donor cells in nuclear transfer in the later study.
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