P190-Rho_GAP(P190) is a member of the Rho family GTPase activating proteins, that have been shown to play a role in cytokinesis and play roles in cell proliferation. Studies have shown that p190 plays various roles in nervous system development and defects are similar to defects seen in cell adhesion disorders. In conjunction with another research project examining the role of p190RHoGAP in jaw development via alcian blue staining, this study set out to optimize genotypic verification of P190 in zebrafish embryos. We used polymerase chain reaction (PCR) to amplify the p190 genetic contributions in individual embryos followed by Restriction Fragment Length polymorphism (RFLP) analysis to distinguish between different genotypes of the P190 gene; wild type(250 and 350b.p), mutant (600b.p), heterozygous (600, 350 and 250 b.p). Currently the common methods include single cell extraction at the 32-cell stage or head vs. tail dissections which are both technically challenging. While head vs tails extractions have been previously utilized in our lab, we sought to streamline this process to exclude embryo dissections. During the procedure, PCR optimization and trouble shooting had to be done prior to, determining if DNA samples would be viable for PCR when DNA extractions were done on previously dyed embryos . As a control we compare previously dyed embryos to tailed embryos that were non-dyed tail extractions. Our results indicate the alcian blue staining did not have a notable impact on our genotypic verification when comparing the stained and non-stained embryos. Our data highlight the delicate nature of working with PCR on different embryo treatments which can halt forward progress in genotypic analysis of mutant embryos that may have subtle or late developing phenotypes.
Saucedo, Brian Gurrola and James, Andrea, "Adventures in PCR; the Impact of Alcian Blue Staining on Danio rerio Embryos" (2020). 2020 Undergraduate Presentations. 11.