International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)


D7. A Novel Method for Constructing Gene-Targeting Vectors

Kiyotaka Akiyama, Hirotaka Watanabe, Shuichi Tsukada, and Hitoshi Sasai
Pharmaceutical Frontier Research Laboratory, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 13-2, Fukuura 1-chome, Kanazawa-ku, Yokohama, Kanagawa, Japan

We developed a simple and rapid method of constructing knockout vectors using inverse-PCR (IPCR). The method consists of 3 steps: 1) digestion of a target bacterial artificial chromosome (BAC) with several restriction enzymes (six-base cutters) followed by self-ligation, 2) IPCR using circular DNAs as templates and two primers which are oriented in the reverse direction and 3) cloning into a vector containing a positive selection marker, which results in a typical replacement knockout vector. We successfully targeted 3 mouse genes including the HPRT gene by using this method. Compared with the conventional method, this method has several advantages. First, it is relatively quick. While the conventional method requires several months for completion of a targeting vector, this novel method requires no more than 3 weeks. Second, this method requires much less sequence information about the target than does the conventional method. So we can start with only a small piece of information such as that EST. Third, this method makes it easy to insert any sequence into any desirable region in a target gene. This can overcome the difficult that site-directed mutation or deletion is often impossible with conventional method due to the limited availability of preferred restriction site. We believe that this method allow us an efficient production of knockout mice.


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