Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Kršnjavoga 25, 10000 Zagreb, Croatia.
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
FEMS Yeast Res. 2017 Jun 1;17(4). doi: 10.1093/femsyr/fox041.
Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes.
基因替换是构建基因工程酵母菌株的最基本方法之一。然而，基因靶向的准确性和构建特定菌株所需的努力可能会有很大差异。我们研究了两个重要因素的影响——转化方法的选择和转化 DNA 片段的设计，与基因组中靶向区域的长度相比，转化 DNA 片段的全长（包括侧翼区域和选择性标记）可能会有所不同。我们使用电穿孔和原生质体转化在几个测定中确定了基因替换的准确性，并与我们之前通过锂醋酸盐获得的结果进行了比较。我们清楚地表明，基因靶向准确性取决于转化方案，锂醋酸盐方法的准确性最高。相比之下，电穿孔和原生质体转化的准确性较低。此外，准确性还取决于转化测定的设计，因为转化 DNA 和靶向区域的全长比值越高，准确性越高。此外，通过 qPCR 对异常转化体的核型分析表明，基因靶向可能导致单倍体菌株的二倍化，最有可能通过靶向染色体复制，随后复制其他染色体。
