I have been trying to rescue a gene knock out phenotype by complementing with the same gene under a plasmid, but the rescue I am getting is close to 30% . I have tried differential promoter strengths including native promoter to enhance the rescue but no significant improvement in rescue was observed.
what could be the underlying problem? Using S.cerevisiae as model.
Tricky question, as there are many elements that may influence the strength of your rescue, for example elements in the 3'UTR of the gene that are not included in your rescue construct.
What is the exact composition of your rescue construct? Are you using gDNA or cDNA? Extra regulatory information may be situated in the introns as well.
Ideally, you could try rescuing with a large genomic clone that consists of a large upstream/downstream region, and all intronic data of your gene of interest.
There's always the possibility of a background mutation affecting phenotype you are screening for, and that that is leading to only partial rescue. Maybe you can try rescuing different, independently generated knockout genes?
(I should note that my experience in rescuing phenotypes comes from C. elegans and not yeast.)
Hope that helps.
Try to characterize the amount and/or post-translational modifications of the protein produced from the plasmid. If it is equivalent to that found in the wild type, then the experiment is giving you the information it is supposed to provide -that the phenotype of the knocked-out cells is not due solely to the knockout.
Hope it helps!
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