Abstract P095: An Expression-Based in Vivo Protein Trap Screen Enriches Cardiac Mutants and Reveals Cardiac Functions of Methionine Adenosyltransferase 2A in Zebrafish
The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based in vivo protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines, RP2_ #135, is a homozygous embryonic lethal line that is caused by a RP2 insertion in methionine adenosyltransferase 2a (mat2a). Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in RP2_ #135 is due to a disrupted expression of mat2a in cardiomyocytes, as indicated by a conditional rescue assay using Tg(cmlc2:Cre-YFP), a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the mat2a mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.
- © 2011 by American Heart Association, Inc.