Generation and characterization of the bmp16-null knockout zebrafish line. (A) Whole-mount in situ hybridization showing bmp16 expression in wild-type zebrafish embryos at the indicated stages. Red triangles mark the embryonic hearts. Scale bars: 200 μm. (B) Schematic of the CRISPR/Cas9 targeting strategy to generate the bmp16 knockout. Open and filled boxes represent untranslated regions (UTRs) and coding sequences, respectively. Purple bars indicate guide RNA binding sites; arrows denote primer binding sites for genotyping. (C) Genomic sequences of wild-type (WT) and bmp16 knockout (KO) alleles at the gRNA target sites. gRNA target sequences are shown in purple, with the protospacer adjacent motif (PAM) sequences in green. The coding sequences are capitalized, with the signal peptide-coding sequences underscored. (D) Representative genotyping results of wild-type (bmp16+/+), heterozygous (bmp16+/−), and homozygous (bmp16−/−) embryos. (E) WISH analysis of bmp16 expression in wild-type and bmp16 knockout zebrafish embryos at 72 hpf. Scale bar: 200 µm. (F) Quantitative RT-PCR analysis of bmp16 expression at 24 hpf in the indicated genotypes. Statistical analysis was performed using one-way ANOVA. (G–I) Quantitative expression levels of BMP target genes id1 (G), id2a (H) and id2b (I) in wild-type and bmp16 knockout zebrafish embryos. The statistical analyses were performed using unpaired Student’s t-tests. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.
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