Cre-recombinase restores wild type expression of grin2bbART in bigheart mutant. (A)Expression profiling of grin2bbART transcript in adult zebrafish using whole mount in situ hybridization(B)grin2bbART expression in bh−/− heart tissue (A: atrium; V: ventricle; BA: bulbus arteriosus) (C) qRT PCR analysis showing the relative expression of grin2bbART in WT and bh−/− heart tissues. (D) Schematic depicting Cre-recombinase mediated gene trap reversion in bigheart mutant. Triangles in pink represent the loxP sites flanking the gene trap. By supplying Cre recombinase mRNA, the mutagenicity cassettes and 3′ exon trap are excised. (E)Cre recombinase reverts the bigheart phenotype to wild type, approximately in 50% of the injected embryos, compared to non-injected control. (F) Relative expression of grin2bbART in Cre recombinase injected WT and bh−/− embryos. Black arrowhead shows positions of primers of the PCR analysis of Cre recombinase injected and non-injected bh−/− embryos. N = 5 heart samples were analyzed in each group for the in-situ experiment. The RT-PCR experiments were repeated three times in triplicate reactions. (**, p < 0.01). The bars indicate the average values ± SD.
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