Improvement of Targeting Efficiency by Promoter Replacement of Markers in Integration Vectors

To establish a gene expression system that reflects physiological conditions, we developed a series of vectors that can be integrated into the chromosome. Compared with the integration vectors employing double-crossover recombination, single-crossover integration vectors have the advantage of high transformation efficiency. However, because single-crossover recombination generates repeat sequences upstream and downstream of the integrated fragment, this strategy is often associated with a risk that an integrated fragment may pop out from the chromosome during cultivation. Here, we assessed the frequency of pop-out using a fission yeast single-crossover integration vector, pDUAL. We also examined the effect of shortening the repeats on pop-out by employing a strategy involving heterologous replacement of the promoter for the leu1 marker in the vector. Due to the intrinsic low frequency of pop-out, the effect of promoter conversion on pop-out was negligible, if any. However, a clear ameliorative effect was observed in obtaining the desirable transformants in which a vector fragment was correctly inserted at the targeted locus, a result that may be driven by the limited potential for recombination in the promoter replacement construct.