| 摘要: |
| 【目的】筛选α-1,3半乳糖苷转移酶(α1,3 galactosyl 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。 |
| 关键词: 五指山小型猪 基因缺失 GGTA1基因 成纤维细胞 |
| DOI： |
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| 基金项目: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) |
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| Efficient screen of porcine fibroblasts with GGTA1 gene deficiency (GGTA1-/-) using αGal epitopes difference |
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YUE Cheng-he,CAO Sui-zhong,LI Xi-rui,et al
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| Abstract: |
| 【Objective】The study aimed to screen fibroblasts with deficiency of α1,3 galactosyl transferase gene(GGTA-1).【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-B-4.Magnetic activated cell sorting (MACS),combination of MACS with fluorescence-activated cell sorting (FACS),and combination of Clostridium difficile Toxin A (TcdA) with FACS methods 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 identified 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%. 【Conclusion】MACS and combination of TcdA with FACS could efficiently screen WZSP fibroblasts with deficiency of GGTA1 gene.This study would improve further study on genetic modification of pigs for xenotransplantation. |
| Key words: Wuzhishan miniature pigs (WZSP) gene deficient α1,3 galactosyl transferase gene(GGTA 1) fibroblasts |
