A novel method for constructing gene-targeting vectors.
We developed a simple and rapid method for constructing knockout vectors using inverse-PCR (IPCR). The method consists of three steps: (i) digestion of a target bacterial artificial chromosome with several restriction enzymes (six-base cutters) followed by self-ligation; (ii) IPCR using circular DNAs as templates and two primers which are oriented in opposite directions; and (iii) cloning into a vector containing a positive selection marker, which results in a typical replacement knockout vector. We successfully targeted three mouse genes including the HPRT gene using this method. Compared with the conventional method, this method requires much less time (no more than 3 weeks). Notably, this method requires only small amounts of sequence information (several hundred base pairs such as is available from expressed sequence tags) and can be extended to a systematic mass production of targeting vectors applicable to many organisms, including yeast.
Akiyama
K
K
Pharmaceutical Frontier Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 13-2, Fukuura 1-chome, kanazawa-ku, Yokohama, Kanagawa, Japan. kiyotaka.akiyama@ims.jti.co.jp
Watanabe
H
H
Tsukada
S
S
Sasai
H
H
D000818:Animals; D002460:Cell Line; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D020224:Expressed Sequence Tags; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction / Q000379:methods*; D017467:Receptors, Erythropoietin / Q000235:genetics; D014162:Transfection
D004269:DNA, Bacterial; D017467:Receptors, Erythropoietin; D007041:Hypoxanthine Phosphoribosyltransferase