利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪  

Production of GHR double-allelic knockout Bama pig by TALENs and handmade cloning

作　　者：李飞达 李勇 刘欢 张欢欢 刘楚新 张兴举 窦红伟 杨文献 杜玉涛 

机构地区：[1]深圳华大基因研究院,深圳518083 [2]深圳动物基因组辅助育种工程实验室,深圳518083 [3]深圳华大方舟生物技术有限公司,深圳518083

出　　处：《遗传》2014年第9期903-911,共9页Hereditas（Beijing)

基　　金：农业部动物转基因重大专项课题(编号:2014ZX0801007B);深圳市生物产业发展专项资金(编号:JC201005260182A)资助

摘　　要：DNA编辑技术是基因靶向修饰技术的研究热点,已广泛应用于生物医学和农业研究。然而,传统基因打靶技术存在效率低、成本高、工作量大等缺点,其应用受到了极大的限制。文章利用最近发展起来的新型人工核酸酶——转录激活因子样效应物核酸酶(Transcription activator-like effector nuclease,TALENs)介导的基因组定点修饰技术,通过构建特异识别猪生长激素受体(GHR)基因的TALENs表达载体,共转染巴马胎猪成纤维细胞系,酶切鉴定G418抗性克隆细胞株的基因修饰效率为46.2%,其中2个为双等位基因敲除的克隆细胞株。以双等位基因敲除的克隆细胞株为核供体,利用手工克隆技术制备GHR-KO巴马猪克隆胚,第6 d囊胚率为43.5%,654枚体外发育的囊胚移植6头受体母猪,共获得10头存活的GHR-KO巴马仔猪,其中7头为双等位基因敲除。体重检测结果显示,第20周龄的GHR-KO巴马猪体重仅为对照巴马猪的50%。研究结果表明,TALENs和手工克隆技术能够高效制备出基因敲除大动物。GHR-KO巴马猪的成功制备为研究猪GHR基因生理功能以及人类侏儒症分子机理提供了重要模型,也证明该技术比传统基因打靶和克隆技术具备更简捷、快速、高效,更易于在生物医学和农业基因靶向修饰研究中推广。DNA editing techniques for targeted genome modification have witnessed remarkable advances and been widely used in various organisms.However,traditional gene targeting and cloning method has been shown to be low efficient,time-consuming and expensive for generating knockout animals,especially for big animals.Here we report the generation of site-specific genome modified pig with the newly developed artificially engineered sequence-specific endonucleases （transcription activator-like effector nuclease,TALENs） and handmade cloning （HMC） methods.First,we constructed the porcine GHR-knockout vector according to TALENs kit protocol.To obtain the nuclear donor,the fetal fibroblast cell of Bama （BM） pig were transfected with GHR-knockout vector in G418 selection medium.We collected 173 cell for further positive identification which showed that 46.2% （78/173） of the clones were GHR-knockout cell strains.We chose one bi-allelic knockout cell strain as nuclear donor to produce reconstructed embryos by HMC.It was shown that the blastocyst rate was 43.5% at the 6th day in vitro,then 654 HMC-blastocysts were transplanted to uterus of six recipient sows.Finally,a total of 10 live offspring were delivered including 7 bi-allelic knockout piglets.Fibroblasts were obtained from ear biopsies for GHR knockout detection.The body weight of the piglets was measured consecutively,and it was found that the GHR^-pigs were only 50% smaller than that of the controls at the 20th week.In conclusion,our results indicate that TALENs and HMC technology can rapidly and efficiently produce knockout animals for agricultural and biomedical research.

关 键 词：基因敲除 TALENs 手工克隆 GHR-KO 

分 类 号：Q78[生物学—分子生物学] S828[农业科学—畜牧学]