A PCR-based strategy to generate integrative targeting alleles with large regions of homology
A PCR-based strategy to generate integrative targeting alleles with large regions of homology
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DOI：
10.1099/00221287-148-8-2607
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发表时间：
2002-08-01
期刊：
影响因子：
2.8
通讯作者：
Heitman, J
中科院分区：
文献类型：
--
作者：
Davidson, RC;Blankenship, JR;Heitman, J
新生隐球菌是一种机会性真菌病原体，具有明确的性周期，其遗传和分子技术已经得到了很好的发展。一种新型C菌菌株的整个基因组序列即将完成。这种序列的有效利用依赖于更快速地进行遗传分析的方法的发展，包括基因破坏技术。描述了一种改进的PCR重叠技术，以产生针对包含大范围基因同源区域的基因中断的靶向构建体。这项技术被用来干扰或删除十几个基因，其效率与之前报道的使用克隆技术产生靶向构建体的效率相当。此外，研究表明，该技术可以在各种菌株背景下进行干扰，包括致病A型分离株H99和最近鉴定的稳定的二倍体菌株。结合完整基因组序列的可用性，这种基因破坏技术应该为这种重要的致病真菌的更高吞吐量的遗传分析铺平道路。
Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.