Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans:: Absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes
Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans:: Absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes
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DOI：
10.1016/j.fgb.2006.02.007
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发表时间：
2006-08-01
影响因子：
3
通讯作者：
Lodge, Jennifer K.
中科院分区：
文献类型：
--
作者：
Goins, Chelsey L.;Gerik, Kimberly J.;Lodge, Jennifer K.
新型隐球菌是一种致病性真菌，其相对易于进行分子遗传学分析，包括基因缺失。然而，同源重组的速率可能很低，因此获得特异性基因缺失转化体具有挑战性。我们已经利用了两种新技术，cku缺失菌株，以提高该生物体中基因缺失的效率，和共转化。Ku 70-Ku 80异源二聚体被预测为在新型隐球菌中非同源末端连接过程的重要部分。在这里，我们表明，这些蛋白质中的一个或两个的缺失导致同源重组率的增加。重要的是，我们证明了在产生具有感兴趣的特定缺失的菌株后，可以通过交配和分离去除cku缺失。我们还利用野生型基因和选择标记在单独的线性DNA分子上的共转化来补充缺失事件。我们表明，共转化的结果在野生型表型的成功恢复，虽然这种表型的变化经常发生。(c)2006年爱思唯尔公司All rights reserved.
Cryptococcus neoformans is a pathogenic fungus that is relatively amenable to molecular genetic analysis, including gene deletion. However, rates of homologous recombination can be low, so obtaining specific gene deletion transformants is challenging. We have utilized two new technologies, cku deletion strains to improve the efficiency of gene deletions in this organism, and co-transformations. The Ku70-Ku80 heterodimer is predicted to be an essential part of the non-homologous end-joining process in C neoformans. Here we show that a deletion in one or both of these proteins results in an increase in the rates of homologous recombination. Importantly, we demonstrate that after generation of a strain with a particular deletion of interest, the cku deletion can be removed by mating and segregation. We also utilize co-transformation of wild-type genes and selectable markers on separate linear DNA molecules to complement a deletion event. We show that co-transformation results in the successful restoration of wild-type phenotype, though variations in this phenotype often occur. (c) 2006 Elsevier Inc. All rights reserved.