Study Of Goat Mammary Gland Epithelial Cells Transgected Human β Defensin 3 Gene

The somatic cell nuclear transfer (SCNT) has widely applled for successful production of transgenic animals. Which as the target cells of bioreactor, mammary epithelial cells could secrete the active foreign proteins by the modification and translation of exogenous gene. So it is a effective approach to detect the ablilty of mammary gland specific expression vector by transfected mammary epithelial cells. In this study we systematic discuss the hBD3 gene expression vector transfected mammary epithelial cells in the key procedures. Primary mammary epithelial cells of diary goat were cultivated successfully using tissue mass inoculation method, the optimal parameters for transfection of goat mammary epithelial cells was obtained by transfecting pEGFP-C1 vector with different approach. With the optimal parameters, we obtained stably transfected goat mammary epithelial cells which was determined following G418 selection with transfecting hBD3 gene expression vector, we got goat mammary epithelial clonies which integrated the exogenous gene and expressed the exogenous human-beta-defensin-3 proteins stably by PCR, southern blot and western blot. Following results have been obtained:1 The materials were obtained form the healthy dairy goat of different sources, and the mammary epithelial cells were cultivated successfully by tissue mass inoculation method. There were lots of fibroblasts graw with the primary mammary epithelial cells, and which was removed by using the cell scraper and digesting with trypsin (0.25% trypsin /0.04%EDTA). Finally, we got two groups of purified goat mammary epithelial cells.2 We gained the goat mammary epithelial cells which had the typical features of mammary epithelial cells as cobblestone-like cells. And they were identified by using immunofluorescence staining of cytokeratin 18 and by detecting the growth curve. The results showed that the cells could be stained by cytokeratin 18 and the cells'growth curve similared to the"S", so the cells obtained were mammary epithelial cells and could be used to the following experiments.3 We compared the effectiveness of using lipofection with the different concentrations of DNA (2, 4, 8, 10, 12 ug), different incubation time (2, 4, 6, 10 h) and two different cell lines (GMEC3, GMEC4) of the first generation, 3rd generation, 5th generation, 9th generation. We also compared the effectiveness of using electroporation with the different voltage (120, 140, 160, 180, 200 V), different electric pulse length (5, 10, 15, 20 ms), and two different cell lines (GMEC3, GMEC4) of the first generation, 3rd generation, 5th generation, 9th generation. Finally, we found that with the electroporation conditions, specifically, one 15 ms pulse of 180 V could induce DNA introgression of the mammary epithelial cells effectively. These results all inferred that passage number exert remarkable influence on transfection efficiency than genetic background, the low-passage mammary epithelial cells had the highest transfection efficiency.4 We transfected the hBD3 gene expression vector into goat mammary epithelial cells, and got monoclonal cells which could expressed the EGFP obviously following G418 selection. In our experiments, we identified the integration of the exogenous gene by PCR and southern blot, and we also identified the expression of protein by western blot .The result showed that we obtained the cells which could express the exogenous human-beta-defensin-3 gene stably with the optimal parameters. This study provided the optimal parameters for introducing DNA into mammary epithelial cells and could improve the efficiency of transgenic cloning by SCNT.