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.
Keywords: aneuploidy; diploidisation; gene targeting fidelity; off-targeting events; targeted chromosome duplication; yeast transformation.
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