A rapid and simple method for inactivating chromosomal genes in Yersinia

被引:206
作者
Derbise, A
Lesic, B
Dacheux, D
Ghigo, JM
Carniel, E
机构
[1] Inst Pasteur, Unite Bacteriol Mol & Med, Lab Yersinia, F-75724 Paris 15, France
[2] Inst Pasteur, Grp Genet Biofilms, Paris, France
来源
FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY | 2003年 / 38卷 / 02期
关键词
allelic exchange; Yersinia; mutagenesis; pKOBEG;
D O I
10.1016/S0928-8244(03)00181-0
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:113 / 116
页数:4
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