Single-copy insertion of transgenes in Caenorhabditis elegans.
Single-copy insertion of transgenes in Caenorhabditis elegans.
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DOI：
10.1038/ng.248
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发表时间：
2008-11
期刊：
影响因子：
30.8
通讯作者：
Jorgensen, Erik M.
中科院分区：
文献类型：
--
作者：
Frokjaer-Jensen, Christian;Davis, M. Wayne;Hopkins, Christopher E.;Newman, Blake J.;Thummel, Jason M.;Olesen, Soren-Peter;Grunnet, Morten;Jorgensen, Erik M.
目前，秀丽隐杆线虫的转基因是通过向生殖系注入DNA来产生的。DNA组装成半稳定的染色体外阵列，由注射DNA的许多拷贝组成。这些转基因通常在体细胞中过度表达，而在种系中沉默。我们开发了一种称为MosSCI （mos1介导的单拷贝插入）的方法，将转基因的单个拷贝插入到定义的位点。Mos1转座子的动员在非编码DNA中产生双链断裂。通过从染色体外模板复制DNA到染色体位点来修复断裂。通过注射大约20只动物，可以在不到两周的时间内获得纯合子单拷贝插入。我们成功地插入了长达9 kb的转基因，并证实了在目标位点插入了单个拷贝。单拷贝转基因在内源性水平上表达，可以在雌性和雄性种系中表达。
Currently transgenes in C. elegans are generated by injecting DNA into the germline. The DNA assembles into a semi-stable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method called MosSCI (Mos1-mediated Single Copy Insertion) that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double strand break in non-coding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single copy insertions can be obtained in less than two weeks by injecting approximately twenty animals. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
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