Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.
Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.
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DOI：
10.1186/1471-2164-9-460
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发表时间：
2008-10-07
期刊：
影响因子：
4.4
通讯作者：
Cuppen E
中科院分区：
文献类型：
--
作者：
van Boxtel R;Toonen PW;Verheul M;van Roekel HS;Nijman IJ;Guryev V;Cuppen E
实验鼠(Rattus Norveicus)是生理学和药理学研究中首选的模式生物之一，尽管特定的遗传模型，特别是基因敲除的可用性有限。N-乙基-N-亚硝脲(ENU)驱动的靶向诱变是目前在大鼠体内最成功的方法，但它仍然非常费力和昂贵。由于ENU诱导的DNA损伤通常由错配修复(MMR)系统识别，因此我们假设目标选择突变方法的有效性可以通过MMR缺失的遗传背景来提高。事实上，MSH6基因敲除大鼠被发现对ENU治疗更敏感，生殖系突变率提高了两倍多，达到每585kb 1个突变。此外，分子突变谱被发现改变了~20%，有利于产生敲除型等位基因，导致整体效率提高~2.5倍。在一组只有310只突变的F1大鼠身上，在70Mb的序列中高通量发现了突变，证明了改进的有效性。这导致了89个突变的鉴定，其中4个引入了过早的停止密码子，导致了氨基酸变化。综上所述，我们表明，使用MMR缺陷的背景显著改善了ENU驱动的大鼠目标选择突变，从而减少了动物使用以及筛选成本。使用错配修复缺陷的遗传背景来改进突变和目标选择的敲除效率原则上适用于任何感兴趣的生物体。
The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.
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