Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.
Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.
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DOI：
10.1093/mmy/myu083
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发表时间：
2015-04
期刊：
影响因子：
2.9
通讯作者：
Pavuluri Y
中科院分区：
文献类型：
--
作者：
Lin X;Chacko N;Wang L;Pavuluri Y
新型隐球菌是隐球菌性脑膜炎的病原体，每年导致全世界超过 50 万人死亡。这种荚膜担子菌酵母也可作为微病原研究的模型。通过异位整合或同源重组产生稳定突变体的能力已通过基因枪转化实现。这一技术进步极大地促进了过去二十年来对该病原体的基础生物学和致病机制的研究。然而，生物射弹转化成本高昂，且其重现性差异很大。在这里，我们发现通过电穿孔转化可以实现稳定的异位整合或通过同源替换的靶向基因删除。通过电穿孔获得的转化体的稳定性和同源替换的频率高度依赖于选择标记。当使用显性药物选择标记时，通过电穿孔获得的稳定转化子之间的同源重组频率与通过基因射弹转化获得的同源重组频率相当（~10%），这远高于先前报道的使用营养缺陷型标记时的电穿孔（0.001%至0.1%）。此外，使用分裂标记策略破坏KU80基因或产生基因缺失构建体，这两种已知可增加通过基因枪转化获得的转化体中同源替换的方法，也增加了通过电穿孔获得的转化体中同源替换的频率。因此，电穿孔为隐球菌诱变提供了一种低成本的替代方案。
Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (~10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.