Liu Jirong, Zogaj Xhavit, Barker Jeffrey R, Klose Karl E
University of Texas San Antonio, San Antonio, TX 78249, USA.
Biotechniques. 2007 Oct;43(4):487-90, 492. doi: 10.2144/000112574.
Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.
土拉弗朗西斯菌是最致命的细菌病原体之一，然而其发病机制的大多数遗传决定因素仍不清楚。我们通过利用优化的抗生素抗性盒的通用引物和重叠延伸剪接（SOE），在模式生物土拉弗朗西斯菌新凶手亚种中开发了一种高效的靶向诱变策略。这一过程能够在土拉弗朗西斯菌新凶手亚种中快速高效地构建具有非极性突变特征的靶向插入突变；优化的靶向诱变策略将促进对这种神秘细菌的研究，并有助于开发抗兔热病疫苗。此外，基于PCR的抗生素抗性盒插入进行基因破坏的一般策略广泛适用于许多细菌物种。
