engleski

Construction of yeast strains - fun and easy?!

Gene targeting is a genetic technique that allows modification of an endogenous gene by homologous recombination. This is usually achieved by introduction of linear, non- replicative DNA fragments that should replace homologous (targeted) sequence in the genome. While this procedure is widely used for the construction of different strains it is also studied as a model for targeted genetic manipulations by the "ends-out" recombination. It is generally assumed that, in contrast to other yeasts and higher eukaryotes, transformation of the yeast Saccharomyces cerevisiae with non-replicative DNA almost exclusively occurs by homologous recombination. However, we found that even in S. cerevisiae ends-out recombination may result in off- targeting events. These events include different genome alterations such as illegitimate integration, integration of the transforming DNA fragment next to the target region and duplication of a targeted chromosome. Therefore, we systematically investigated the influence of a design of the ends-out assay and transformation method on the transformation efficiency, fidelity of gene targeting and spectra of all aberrant events. We show that the fidelity of gene targeting in S. cerevisiae is mainly influenced by the nature of alleles involved in recombination and we propose guidelines that increase the efficiency of gene targeting.

yeast, transformation, gene targeting

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