Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing 100101, People's Republic of China.
J Microbiol Methods. 2009 Dec;79(3):253-9. doi: 10.1016/j.mimet.2009.09.004. Epub 2009 Sep 16.
In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), split marker deletion method, co-transformation of single-stranded DNA and mutant Cre-loxP system. Using a slightly modified LiAc/SS-DNA/PEG procedure, the co-transformation double-stranded split marker constructs together with single-stranded split marker constructs resulted in at least 70% homologous recombination events when the homologous genomic DNA fragment had a size of approximately 500bp. Our evidence suggested that single-stranded DNA may be responsible for the increased gene disruption efficiency. We demonstrated the effectiveness of the method for gene disruption by constructing both single and double gene disruptions at the ALG3 and URA5 loci in the same genetic background. The method described here presents an improved strategy for gene disruption and a potential application for investigation of biological processes in other yeast strains.
在聚球藻（H. polymorpha）中，仍然缺乏一种高效的基因敲除方法。为了帮助解决这个问题，我们提出了一种在聚球藻中进行单基因和多基因敲除的简单而有效的方法。该敲除系统结合了粘性末端聚合酶链反应方法（SEP）的变体、分割标记缺失方法、单链 DNA 的共转化和突变 Cre-loxP 系统。通过略微修改的 LiAc/SS-DNA/PEG 程序，当同源基因组 DNA 片段的大小约为 500bp 时，共转化双链分割标记构建体与单链分割标记构建体一起导致至少 70%的同源重组事件。我们的证据表明，单链 DNA 可能负责提高基因敲除效率。我们通过在相同的遗传背景下在 ALG3 和 URA5 基因座构建单基因和双基因敲除，证明了该方法的有效性。这里描述的方法为基因敲除提供了一种改进的策略，并为研究其他酵母菌株中的生物过程提供了一种潜在的应用。
