利用正负筛选打靶载体在猪肾细胞系PK-15细胞中敲除GGTA1基因  

Disruption of GGTA1 gene with positive-negative targeting vector in porcine kindey cell line PK-15 cells

作　　者：常宏宇[1,2] 宫锋[2] 季守平[2] 鲍国强[2] 李素波[2] 章扬培[2] 李芳[1] 聂亚玲[1] 

机构地区：[1]第二炮兵总医院儿科,北京100088 [2]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学科学院院刊》2010年第3期234-238,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家重点基础研究发展计划(973计划)(2002CB713804)

摘　　要：目的半乳糖-α1,3-半乳糖(αGal)表位是猪到人异种移植中引起排斥反应的主要异种抗原。通过同源重组敲除猪α1,3-半乳糖基转移酶基因可从异种移植物表面彻底清除αGal表位。本研究的目的在于利用正负筛选打靶载体定向缺失猪GGTA1基因。方法构建猪α1,3-半乳糖基转移酶基因正负筛选打靶载体pSL/GT,载体包含了正负筛选标记基因neo和tk,打靶载体线性化后用脂质体包裹转染猪肾细胞系PK-15细胞,利用G418和GANC进行抗性细胞克隆的药物筛选,挑选抗性克隆进行PCR及Southern杂交鉴定。结果共得到436个抗性细胞克隆,其中31个克隆PCR结果阳性。经Southern杂交鉴定证实,其中1个克隆发生了同源重组,为杂合子。结论本研究构建了有效的猪GGTA1基因的正负筛选打靶载体pSL/GT,并利用其在猪肾细胞系PK-15细胞中定向缺失GGTA1基因,获得了杂合子GGTA1(+/-)PK-15细胞。Objective The epitope galactose-α1,3-galactose（αGal）is the major xenoantigen causing rejection in pig-to-human xenotransplantation.Disruption of the gene encoding pig α1,3-galactosyltransferase（GGTA1 or α1,3GT）by homologous recombination is a means by which to completely remove the αGal epitopes from the xenografts.The objective of this study was to disrupt GGTA1 gene with a positive-negative targeting vector in porcine kindey cell line PK-15 cells.Methods The gene targeting vector pSL/GT,which included the positive selection marker neo and negative selection marker tk,was constructed.PK-15 cells were transfected with linear pSL/GT using LipofectamineTM 2000.The transfected cells were cultured for 48 hours without selection before the drug G418（800 μg/ml）and GANC（1.5 μg/ml）were added.After 10-12 days of selection,the resistant cell clones were isolated and then identified by PCR and Southern blot.Results Totally 436 resistant PK-15 cell colonies were picked up after being cultured in G418 and GANC.Of these resistant cell colonies,31 were positive according to PCR and only one was GGTA1（＋/-）cell according to Southern blot.Conclusion An effective porcine GGTA1 gene targeting vector pSL/GT with positive-negative markers was constructed,and heterozygous GGTA1（＋/-）PK-15 cells were obtained.

关 键 词：猪 α-Gal表位 Α1 3-半乳糖基转移酶 基因打靶 异种移植 

分 类 号：Q78[生物学—分子生物学]