线虫基因显微注射和整合技术——设备、操作与指南
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国家自然科学基金资助项目(30670502, 30500117).


Gene Microinjection and Integration in C. elegans: Equipments, Processes and Guidance
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This work was supported by a grant from The National Natural Science Foundation of China (30670502, 30500117).

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    摘要:

    秀丽线虫(Caenorhabditis elegans)是研究动物遗传、个体发育和行为活动的重要模式生物,其主要优点是能够研究从分子细胞水平到整体系统水平的相关生命活动的作用机理.其中基因显微注射和整合是该领域的核心技术,广泛应用于研究线虫的基因表达、功能和基因间的相互作用等.显微注射技术使用尖端开口直径为微米级的玻璃微管,将所研究的目的DNA注射进线虫的性腺.注射的DNA被成熟的卵细胞吸收,以染色体外遗传物质的形式存在.但这种形式并不能稳定遗传,可采用基因整合技术将外源基因整合到染色体上,得到稳定遗传的线虫种系.显微注射是一项精细的实验技术,影响实验成功与否的因素较多,实际操作需要有一定的经验.在实践过程中逐步改进和完善了这项技术,在此主要介绍线虫显微注射技术的操作过程、创新方法以及注意细节.

    Abstract:

    Caenorhabditis elegans is one kind of the most important modal animals for studying heredity, development and behaviors, and for investigating the mechanisms underlying from molecular to systematic levels. Gene microinjection and integration are the most important ways to make transgenic animals in C. elegans. They are widely used in the studies of expression, function and interaction of genes. Briefly, a DNA construct (plasmid or cosmid) or PCR product which carried gene(s) of interest is mixed with a co-injection marker and injected into the distal gonad (syncytium) through a micropipette. The DNA injected is taken up into the mature oocytes and exists as an unintegrated extrachromosomal array, which segregates randomly and can be lost. The extrachromosomal arrays are integrated optionally into chromosomes by irradiation of γ rays, X rays, or by TMP/UV integration method. Here, the detail processes and key points of microinjection and gene integration in C. elegans was introduced and the know-how in these techniques was offered.

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酒亚明,张蓉颖,吴政星.线虫基因显微注射和整合技术——设备、操作与指南[J].生物化学与生物物理进展,2009,36(5):648-652

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  • 收稿日期:2008-08-31
  • 最后修改日期:2008-10-31
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  • 在线发布日期: 2008-11-10
  • 出版日期: 2009-05-20