Optimization Of The Transferring Condition Of Human Serum Albumin Gene To Ear Fibeoblast Cell In Yanbian Dairy Goat

In this study, used the specificity gene expression vector of mammary glands—pBCl-HSA contains HSA and Neo gene to transfect ear fibroblasts of Yanbian dairy goat. Then screening transgenic cells with G418, continue to develop and extend, then PCR identification, Make sure the purpose gene is integrated successfully. Due to different cell HSA various condition parameters with liposome transfection, in order to obtain the best transfection efficiency, the experiment used three aspects of the amount of plasmid DNA, the amount of liposome and the time of transfection to optimize the transfection conditions. Finally, analysis transgenic effect of the fibroblasts before and after transfection by cell growth curve and karyotype. Get the following results:1, through the tissue culture method, we obtain the ear fibroblasts of Yanbian dairy goat successfully. Observation of cell morphology and growth rate, then analysis the growth curve and karyotype of the cell, found that4-13generations cell is the best, can be used for subsequent transgene experiment.2, The minimum lethal concentration of G418on ear fibroblasts of Yanbian dairy goat is600μg/ml, screening concentration is700μg/ml, maintain screening concentration is350μg/ml.3, Suitable generation of fibroblasts, transfect pBC1-HSA with LipofectamineTM2000, for24hole cell culture plate, the best amount of plasmid DNA is0.8μg, the amount of LipofectamineTM2000is2.0μl, the optimal transfection time is6h, the highest transfection efficiency is12.27%.4, Extracting the DNA of transgenic cells, then PCR identification, obtain the expectant strip of the purpose gene. So we can confirm that the human serum albumin gene has been integrated into the chromosome genomes successfully.