Cre/Loxp系统介导转基因山羊乳腺上皮细胞标记基因的删除

doi:10.13560/j.cnki.biotech.bull.1985.2019-0544

摘要/Abstract

摘要： 为了消除核移植转基因动物中标记基因潜在的生物安全风险,应用Cre/Loxp系统删除人乳铁蛋白（hLF）转基因山羊乳腺上皮细胞的标记基因,并以此探索标记基因删除前后对功能基因乳腺特异性表达的影响。首先复苏hLF转基因山羊乳腺上皮细胞并纯化;然后电转染PBS185纯化质粒,培养10-14 d,胰蛋白酶消化细胞,通过自制口控式移卵针挑取单克隆细胞;最后PCR检测标记基因是否删除,催乳素诱导表达,ELISA和Western Blot检测功能蛋白hLF的表达情况。结果表明,共获得65株单克隆乳腺上皮细胞,挑取状态较好的18株用于PCR检测,有3株为删除标记基因的细胞株,删除效率达16.7%（3/18）,且删除标记基因细胞株表达水平提高约8倍（2.85 g·L^-1/ 0.35 g·L^-1）,蛋白条带大小与目标蛋白一致（80 kD）。结果表明Cre/Loxp系统能够有效的删除乳腺上皮细胞基因组上的标记基因,且删除后细胞株表达水平明显提高,成功建立一套转基因山羊乳腺上皮细胞标记基因删除的系统,为进一步扩大生产无标记基因的转基因山羊奠定基础。

关键词: 标记基因, Cre/Loxp, hLF, 乳腺上皮细胞, 诱导

Abstract: In order to eliminate the potential biosafety risk of marker genes in transgenic animals by nuclear transfer,the Cre/Loxp system was used to delete the marker genes of the transgenic goat mammary epithelial cells（GMECs）with human lactoferrin（hLF）,so as to explore the effect of marker gene deletion on the mammary gland specific expression of functional genes. First,the transgenic hLF goat mammary epithelial cells were revived and purified. Then,the purified PBS185 plasmid was electrotransfected into mammary epithelial cells. After 10-14 d culture,the monoclonal cells were selected by self-made mouth-controlled transfer needle and trypsinase digested cells. Finally,whether or not the deletion of the marker gene was detected by PCR,prolactin nduced expression and the expression level of functional protein hLF was detected by ELISA and Western Blot. The results showed that a total of 65 monoclonal mammary epithelial cells were obtained after electrotransfection,and 18 of the cells in fine condition were detected by PCR. Among them,3 cell lines were deleted marker gene,and the deletion efficiency was 16.7%（3/18）. Moreover,the expression level of marker gene-deleted cell lines increased about 8 times （2.85 g·L^-1/0.35 g·L^-1）,the size of protein band was consistent with the target protein（80 kD）. The above results prove that the Cre/Loxp system can effectively delete the marker gene in GMECs genome and the expression level of cell lines significantly increase. Moreover,a system for the deletion of the marker gene in transgenic GMECs is successfully established,which also lays a foundation for the further expanding the production of transgenic goats without the marker gene.

Key words: marker gene, Cre/Loxp, hLF, mammary epithelial cells, induction

宋绍征, 于康英, 陆睿, 张婷, 陈朝军, 潘生强, 成勇, 周鸣鸣. Cre/Loxp系统介导转基因山羊乳腺上皮细胞标记基因的删除[J]. 生物技术通报, 2019, 35(12): 105-111.

SONG Shao-zheng, YU Kang-ying, LU Rui, ZHANG Ting, CHEN Chao-jun, PAN Sheng-qiang, CHENG Yong, ZHOU Ming-ming. Deletion of the Marker Gene in Transgenic Goat Mammary Epithelial Cells by Cre/Loxp[J]. Biotechnology Bulletin, 2019, 35(12): 105-111.

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