Abstract
The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.
Highlights
The yeast S. cerevisiae is a popular model organism for studying gene function in eukaryotes
In order to delete the gene of interest conditionally, this gene, along with its promoter and terminator regions, were inserted into the multi-cloning site (MCS) sites flanked by the Flp recombinase recognition target (FRT) target sequence of Flp recombinase
The MCS flanked by FRT sites was inserted into centromeric plasmid pRS314, which served as the backbone for the yeast shuttle vector
Summary
The yeast S. cerevisiae is a popular model organism for studying gene function in eukaryotes. Many techniques have been developed to study the functions of endogenous or exogenous genes in yeast. To regulate gene expression in yeast, genes are generally placed under the well-characterized inducible or repressible (regulatable) promoters. Used inducible promoters include the yeast endogenous GAL1, GAL10, CUP1 and MAL62. MAL62 promoter is induced by maltose and repressed by glucose (Finley et al, 2002). The repressible promoters, such as yeast endogenous MET3 and MET25 (Mumberg et al, 1994), can be repressed in the presence of methionine. The yeast PHO5 promoter is negatively regulated by inorganic phosphate (Rogers et al, 1982). There are the Tet-off promoters, which were adapted
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