We are trying to make conditional mutants in essential genes of Schizosaccharomyces pombe, using the nmt1 promoter and different degrons, and antibiotic selection in haploid cells. The homologous recombination works and we get the expected insertions, but the lack of phenotypes has prompted us to check whether the genes were actually interrupted... and they are, but it turns out that a wt copy duplicates elsewhere in the genome (which explains why there is no phenotype).
In diploid cells, we don't obtain any positives after selection.
Could anyone give me insight about reasons for these duplications, and how to avoid this problem?
Best,
Ruben
I guess (without being an expert) that the answer is in your question - the gene you are targeting is essential. You are applying selective pressure - either cell die or duplicate this gene elsewhere. my2c
Indeed, there has to be selective pressure. However they should be conditionally turned off, so unless they are required during recombination that selective pressure shouldn't apply. Also, pombe cells are in 2c state ~90% of their cycle, so if one chromatid is recombining the other one could still have an active gene... at least in some cells.
But selective pressure being a problem is a very good thought out of the box, definitely.
I agree with Petre Ianakiev. I had similar problems with essential gene in Saccharomyces cerevisiae and found out that this problem can be strain specific (different genetic backgrounds in different strains). I would suggest you to try different strain.
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