利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪

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

doi:10.3724/SP.J.1005.2014.0903

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2014 , Vol. 36 >Issue 9: 903 - 911

DOI: https://doi.org/10.3724/SP.J.1005.2014.0903

研究报告

利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪

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

李飞达,硕士,助理研究员,专业方向：动物基因工程。

收稿日期: 2014-02-27

网络出版日期: 2014-09-20

基金资助

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

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

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1. BGI-Shenzhen, Shenzhen 518083, China; 2. BGI ARK Biotechnology Co., Ltd, Shenzhen 518083, China; 3. Shenzhen Engineering Laboratory for Genomics-Assisted Animal Breeding, Shenzhen 518083, China

Received date: 2014-02-27

Online published: 2014-09-20

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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。

关键词： 基因敲除; TALENs; 手工克隆; GHR-KO

本文引用格式

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 . 利用TALENs和手工克隆技术高效获得GHR基因敲除巴马猪[J]. 遗传, 2014 , 36(9) : 903 -911 . DOI: 10.3724/SP.J.1005.2014.0903

Abstract

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 6^th 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 20^th week. In conclusion, our results indicate that TALENs and HMC technology can rapidly and efficiently produce knockout animals for agricultural and biomedical research.

Key words： gene knockout; TALENs; handmade cloning; GHR-KO

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