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Attempts to integrate a transgene into a specific locus in the mouse genome by microinjection to fertilized eggs

尝试通过受精卵显微注射将转基因整合到小鼠基因组的特定位点

基本信息

批准号：
12558095
负责人：
ARAKI Kimi
金额：
$ 8.51万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558095/
关键词：
Cre lox system Microinjection Tranegenic mice Site-specific integration

项目摘要

In order to integrate a transgene into a specific locus in the mouse genome by microinjection, an efficient site-specific recombination system is indispensable. We have developed Cre-mutated lox system to promote integrative reaction by Cre recombinase. We used heterospesific lox sites that do not recombine with the wild type loxP but itself. Two lox sequences, lox511 and lox2272, were compared with their recombination efficiencies in ES cells. Three cell lines carrying a construct of lox71-bsr gene-lox511-lox2272 were established, and introduced the Cre-expression vector and the replacement vector, which is lox66-LacZ-lox511 and/or lox2272, by electoporation. Since lox71, loxSll and lox2272 are recombined only with lox66, lox511 and Iox2272 respectively, thebsr gene on the ES genome is replaced by the LacZ gene through Cre-mediated recombination. We found that the combination of lox66/71 and lox2272 gave the best efficiency. Then, we tried to establish Cre-mediated site-directed integration system by microinjection into fertilized eggs. Transgenic mouse lines were produced with the transgene of CAG promoter-EGFP-lox71-Puro-pA-lox511-lox2272. The Cre expression vector and the replacement vector, which is lox66-IRES-NLSLacZ-lox511-lox2272, were microinjected into the pronuclei of the fertilized eggs. We examined 85 transgene positive embryos, but no site-directed integration event was detected. Since the transgenic mice carried several copies of the transgene, next, we used three mouse lines which were obtained by gene trapping with the trap vector carrying lox71 and lox2272 and carry single copy of the trap vector pU-17, splice acceptor-lox71-bgeo-loxP-pA-lox2272. About 2000 eggs were injected with the Cre expression vector and the replacement vector containing lox66-EGFP-pA-lox2272, however, only one embryo showed targeted replacement. We are now trying to find optimal conditions of microinjection to improve the efficiency.

为了通过显微注射将转基因整合到小鼠基因组的特定位点，一种高效的位点特异性重组系统是必不可少的。我们开发了一种Cre突变的lox系统来促进Cre重组酶的整合反应。我们使用了异交loxP位点，这些位点不与野生型loxP重组，而是与loxP自身重组。比较了两个lox序列lox511和lox2272在ES细胞中的重组效率。建立了3株携带lox71-bsr基因-lox511-lox2272构建体的细胞系，通过电泳法分别导入cre表达载体和lox66-LacZ-lox511和/或lox2272替代载体。由于lox71， loxSll和lox2272分别仅与lox66， lox511和Iox2272重组，因此通过cre介导的重组，ES基因组上的bsr基因被LacZ基因取代。结果表明，lox66/71与lox2272的组合效率最高。然后，我们尝试通过微量注射到受精卵中建立cre介导的位点定向整合系统。用CAG启动子egfp -lox71- puro - pa -lox511-lox2272转基因小鼠。将Cre表达载体和替代载体lox66-IRES-NLSLacZ-lox511-lox2272微注射到受精卵原核中。我们检测了85个转基因阳性胚胎，但没有检测到位点定向整合事件。由于转基因小鼠携带了多个拷贝的转基因基因，接下来，我们使用了用携带lox71和lox2272的诱捕载体和携带单个拷贝的pU-17（剪接受体-lox71-bgeo- loxp - pa -lox2272）诱捕获得的3个小鼠系。将Cre表达载体和含有lox66-EGFP-pA-lox2272的替代载体分别注入约2000个卵子，但只有一个胚胎出现了靶向替代。我们目前正在努力寻找最佳的显微注射条件，以提高效率。