Studying a specific monogenetic disease based on mutations in different individual genes. I try to recreate the disease phenotypes of donors in vitro. I knocked out a gene x with above 90% efficiency based on Sanger sequencing before and after differentiation, with also almost absent protein in western blot similarly like the donor. However, the phenotype of the ko cells is identical to the control non ko cells and completely different from the donors according to optimized assays to measure the disease phenotype after differentiation. It is important to note that those assays could detect differences in other KO genes I created of the same disease, giving similar phenotypes as the donors. In addition, the donors mutation are mutations while mine is guiided through multiple guide RNAs, still giving normal readouts. What could be an explanation and what would you do next?
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