中国农业科学 ›› 2014, Vol. 47 ›› Issue (21): 4211-4223.doi: 10.3864/j.issn.0578-1752.2014.21.007

• 高效、安全、规模化转基因技术:机会与挑战 • 上一篇    下一篇

规模化转基因技术体系构建及其应用

牟玉莲,阮进学,吴添文,程英,魏景亮,樊俊华,李奎   

  1. 中国农业科学院北京畜牧兽医研究所,北京100193
  • 收稿日期:2014-04-02 修回日期:2014-06-27 出版日期:2014-11-01 发布日期:2014-11-01
  • 通讯作者: 李奎,E-mail:likui@caas.cn
  • 作者简介:牟玉莲,E-mail:muyulian76@iascaas.net.cn
  • 基金资助:
    转基因生物新品种培育重大专项(2014ZX08010-004)、中国农业科学院科技创新工程(ASTIP-IAS05)

Construction and Application of a Scale Transgenic Technology System for Pigs

MU Yu-lian, RUAN Jin-xue, WU Tian-wen, CHENG Ying, WEI Jing-liang, FAN Jun-hua, LI Kui   

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2014-04-02 Revised:2014-06-27 Online:2014-11-01 Published:2014-11-01

摘要: 猪是重要的经济动物,优良猪种的培育对农业的发展具有重要的作用。同时,作为一种常用的试验动物,猪在解剖学、生理学、遗传学等方面与人类高度相似,其在生命科学研究领域中也具有独特的应用价值。转基因技术可以运用基因工程等实验技术手段,对动物基因组进行有目的的遗传修饰,使修饰改造的基因稳定遗传给后代,从而获得满足人类特定需求的动物个体。因此,猪规模化转基因技术体系的建立对转基因育种、基因功能研究和人类疾病模型研究等方面具有重要的意义。本文回顾了国内外猪转基因技术体系的研究进展,总结了目标基因的选择原则,介绍了原核显微注射、体细胞核移植、锌指核酸酶(Zinc-finger nucleases, ZFN)、转录激活子样效应子核酸酶(Transcription activator-like effector nucleases, TALEN)和规律成簇间隔短回文重复序列/ Cas9(Clustered regularly interspaced short palindromic repeat, CRISPR/Cas9)系统、精子载体和逆转录病毒等各种目标基因导入手段,并分析其优缺点和适用范围,列举了表达调控策略中常用的基因调控元件和相关调控系统。近年来,随着转基因重大专项工作的推进,中国猪规模化转基因技术研究取得长足的发展:发掘出大批功能基因;搭建了ZFN、TALEN、CRISPR/Cas9介导的高效基因修饰技术平台及其切割效率检测平台;对供体细胞的分离、培养和筛选流程进行优化;完善体细胞核移植和原核显微注射体系平台,并与新型的基因组编辑技术有机结合,实现目的基因定点、高效、安全的整合;通过将再克隆、胚胎移植、人工受精及常规育种等多种技术的集成创新,建立转基因猪扩繁技术体系,可在较短时间内扩大转基因猪数量;并且实现了这些技术体系的资源共享,为众多单位提供了技术服务支撑,培训技术人员,使转基因技术得到普遍的应用。但是中国猪规模化转基因技术体系仍然存在着一系列的问题:决定转基因猪相关性状并具有自主知识产权的主效基因少;标记基因存在潜在的危害;外源基因随机整合率低、稳定性差;基因打靶等技术环节还不成熟;仔猪成活率低等仍然是猪规模化转基因技术发展面临的瓶颈。因此,要实现猪规模化转基因体系构建的技术性突破,必须结合相关组学工具,深入挖掘并获得具有自主知识产权的重要功能基因和调控元件;构建高效、安全的转多基因载体,实现外源基因的协同、高效表达;完善转基因猪制备过程中的相关技术环节。总之,在猪规模化转基因技术体系的支撑下,中国的转基因猪培育研究必将进入一个新的高速发展时期。

关键词: 猪, 转基因技术, 规模化

Abstract: Swine is an important economic animal and pig breeding plays an important role in the development of agriculture in China. At the same time, as a kind of commonly used experimental animals, pigs are similar in anatomy, physiology and genetics to humans and important for life science research. To meet the specific needs of human beings, animal genomes can be modified by transgenic technology using genetic engineering or other experimental techniques, and the target gene can be passed on to the offspring steadily. Therefore, establishment of a scale swine transgenic technology system is essential for animal breeding, gene function and human disease model research. This paper reviewed the research progress of transgenic swine technology system both in China and foreign countries, summarized the principle of the selection of target genes, described the relative merits and applicable scope of specific gene transfer method such as pronuclear microinjection, somatic cell nuclear transfer, zinc-?nger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/ Cas9), sperm vector and lentiviral vector and so on, expounded the relevant regulatory element and system of gene expression regulation strategy. In recent years, China has made great progress in scale transgenic technology system research since the commencement of National Transgenic Major Projects: including explored a large number of functional genes; built ZFN, TALEN, CRISPR/Cas9 mediated genome modification technology platform; optimized the isolation, culture and screening process of donor cells; improved the somatic cell nuclear transfer and pronuclear microinjection system, and combined them with the novel genome editing techniques for site-specific, safe and efficient integration of exogenous gene; through a further cloning, embryo transfer, artificial insemination and other conventional breeding techniques integrated innovation, finally established the transgenic pig propagation technology system, so that the number of transgenic pigs can be expanded in a short time; provided technical service and trained technical staff for many domestic institutions, made transgenic technology to get a wider range of applications. But a series of problems still exist in the scale swine transgenic technology system of China: the lack of domain genes controlling the transgenic pigs related traits and with independent intellectual property; the transgenic technology supporting system is still not perfect; the potential risks of marker genes; the low integration efficiency and instability of exogenous gene; the inefficiency of gene targeting technology; the low piglets survival rate and so on. All of these are considered to be the bottlenecks of development of scale swine transgenic technology. Therefore, in order to realize the technological breakthrough of scale swine transgenic system, the important functional genes and regulatory element which have independent intellectual property must be explored by related omics tools, construct more efficient, safe and multi-gene transgenic vectors to achieve coordinated and efficient expression of exogenous genes, and improve the related technology in the preparation process of transgenic pigs. In conclusion, the transgenic swine breeding of China will enter a new high-speed development period under the support of scale swine transgenic technology system.

Key words: pig, transgenic system, scale