应用半乳糖表型差异高效筛选α-1,3半乳糖苷转移酶基因缺失的猪成纤维细胞  

Efficient screen of porcine fibroblasts with GGTA1 gene deficiency (GGTA1^(-/-)) using αGal epitopes difference

作　　者：岳成鹤[1,2] 曹随忠[1] 李西睿[2] 冯冲[2] 龙川[2] 余树民[1] 储明星[2] 潘登科[2] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《西北农林科技大学学报（自然科学版）》2014年第1期1-7,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：农业部转基因动物新材料的育种价值评估项目(2011ZX08010-006);中央级公益性科研院所基本科研业务费专项(2012yq-2)

摘　　要：【目的】筛选α-1,3半乳糖苷转移酶(α1,3galactosyl transferase,GGTA1)基因缺失的猪成纤维细胞。【方法】用同源重组载体pBS-GGTA1-SKO和pBS-GGTA1-DKO电转染GGTA1单等位基因敲除(GGTA1+/-)的五指山小型猪胎儿成纤维细胞,利用磁激活细胞分选(MACS)、MACS联合流式细胞术(FACS)、TcdA联合FACS等3种方法进行细胞筛选;对MACS筛选获得的单细胞克隆进行PCR和FACS鉴定,对MACS联合FACS、TcdA联合FACS获得的细胞集落进行PCR鉴定。【结果】经MACS筛选获得了11个细胞克隆,经鉴定2个是GGTA1-/-细胞克隆,阳性率为18.2%;MACS联合FACS筛选获得了3个细胞克隆,经鉴定无GGTA1-/-细胞克隆,阳性率为0;TcdA联合FACS筛选获得了3个细胞克隆,经鉴定均为GGTA1-/-细胞克隆,阳性率为100%。【结论】MACS法筛选和TcdA联合FACS法筛选这2种方法均可以高效筛选出GGTA1双等位基因缺失的猪成纤维细胞,可用于进一步制备供异种器官移植的基因修饰小型猪。[Objective] The study aimed to screen fibroblasts with deficiency of a1,3 galactosyl trans- ferase gene（GGTA1）. [Method] After being transfected with knockout vectors pBS-GGTA1-SKO and pBS-GGTA1-DKO,GGTA1＋/- fetal fibroblasts of Wuzhishan miniature pigs （WZSP） were treated with Phytolectin GSI-B4. Magnetic activated cell sorting （MACS）,combination of MACS with fluorescence-acti- vated cell sorting （FACS）,and combination of Clostridiurn difficile Toxin A （TcdA） with FACS meth- ods were used to screen the cells. Single-cell clones screened by MACS were identified by PCR and FACS. Colonies obtained by combination of MACS with FACS and combination of TcdA with FACS were identi- fied using PCR. [Result] 11 colonies were screened by MACS and 2 of them were identified as GGTA1 / cells with a positive rate of 18.2 %. 3 cell collections were identified by combination of MACS with FACS with a positive rate of 0. Combination of TcdA with FACS obtained 3 cell collections with a positive rate of 100~//00. [Conclusion] MACS and combination of TcdA with FACS could efficiently screen WZSP fibro- blasts with deficiency of GGTA1 gene. This study would improve further study on genetic modification of pigs for xenotransplantation.

关 键 词：五指山小型猪 基因缺失 GGTA1基因 成纤维细胞 

分 类 号：S814.8[农业科学—畜牧学]