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| Construction of Mouse Gene-targeting Vector Through Modified Recombineering Strategy |
| JIANG Li1, YE Xiang-li2, LI Da-li1 |
1. Institute of Biomedical Sciences, East China Normal University, Shanghai 200241, China;
2. College of Medicine, Hunan Normal University, Changsha 410013, China |
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Abstract Gene targeting is a well established technique which allows researchers to create virtually any desired modification in the genome of a living mouse. The first step to generate a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells. Since traditional construction strategy which mainly recruits PCR, restriction digestion and ligation is difficult to get longer DNA fragments for homologous recombination, A strategy using modified recombineering to generate gene targeting vectors was reported. Unlike other methods which use much longer PCR regions for construction, a method used short 50bp homologous regions which were synthesized in PCR primers as the homologous arms to clone more than 10 kb genomic DNA fragment from BAC which contains Gpr56 genomic DNA into a low copy vector through cap repair. Then the 2~5 exon region of Gpr56 was replaced by an ASCI-flanked cassette through a second recmobineering. After that, a DNA fragment which contains a Flp-FRT-based self-excision NEO cassette was then cloned into the vector with ligation through ASCI sites. After DNA sequencing of selected regions, it was confirmed that the Gpr56 targeting vector was what desired.
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Received: 31 March 2011
Published: 25 October 2011
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