[The application of Red/ET recombination to high efficient gene-targeting vector construction].
A rapid and high efficient working system for gene-targeting vector construction was developed by using Red/ET recombination. Mediated by Red/ET recombination, the objective genomic DNA was first subcloned into the targeting vector. After insertion of a PCR amplified selectable marker gene flanked with short homology arms into the targeted position, a conventional gene knock-out targeting vector was then constructed. For conditional gene knock-out targeting vector construction, with the co-operation of Cre-loxP site-specific recombination, two rounds of Red/ET recombination was just needed. Being different from PCR and endonuclease-based gene-targeting vector construction, the homologous regions used for gene targeting can be chosen as long as possible. Furthermore, no enzyme digestion, ligation and sequencing identification were involved, so that it is very efficient and labor-saving. Several different gene-targeting vectors were successfully constructed by using this system. The establishment of this working system will accelerate the gene function studies in the post-genome stage.
Wang
Jun-Ping
JP
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, The Third Military Medical University, Chongqing 400038, China. wangjunp@yahoo.com
Zhang
You-Ming
YM
D003001:Cloning, Molecular; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D011995:Recombination, Genetic
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C084497:RecT protein, E coli; D005090:Exodeoxyribonucleases; C038225:recE protein, E coli