转Temporin-GFP山羊乳腺上皮细胞株的筛选和鉴定

The Screening and Identification of Transgenic Goat Mammary Epithelial Cells with Temporin Gene

为了获得山羊乳腺特异表达中国林蛙抗菌肽的转基因细胞株,研究利用前期工作中获得的山羊乳腺特异表达载体Tem-GFP-pBC1转染传代培养的山羊乳腺上皮细胞,并经G418筛选获得转基因阳性细胞。其结果,获得特异表达GFP和抗菌肽基因的转基因阳性细胞;转基因阳性细胞和冷冻复苏的转基因阳性细胞经培养,其细胞形态和生长曲线均正常;经PCR检测Tem-GFP-pBC1已整合入山羊乳腺上皮细胞的基因组中,且与对照组相比,转基因细胞可以检测到Tem-GFP的表达。结果表明,获得了能够在山羊乳腺中特异表达Tem-GFP融合蛋白的转基因阳性细胞。

In order to obtain Rana temporaria chensinensis skin antimicrobial peptide genes expressed in goat mammary epithelial cells ,goat mammary epithelial cells were transfected with mammary gland specific ex-pression vector Tem-GFP-pBC1 constructed in the previous work by cationic liposome method and cultured with G418-resistant cell .After PCR was identified ,the transgenic cell line with Tem was established .The G418 positive cells were detected .The results were as follows .G418-resistant cells could express GFP and Tem .Mor-phology of the transgenic cells after freezing-thawing was similar to that of normal mammary epithelial cells , the cell growth curve was in“S” shape,PCR results showed that the vector constructed was integrated into ge-nome .Compared to the control ,the transgenic cell could express Tem-GFP .The transgenic cells with temporin-GFP were filtrated ,and lay the foundation for the transgenic goat with expression temporin in mammary .