Global recombination of a floxed tnnt2a allele recapitulates the tnnt2a mutant phenotype. (A) Schematics of the tnnt2a locus showing the positions of the tc300b lesion and of the mn0031 gene-trap cassette; the first LOXP was obtained through Cre recombination of the gene-trap line, and the second LOXP was inserted via CRISPR/Cas9 knock-in using a ssODN donor in the recombined background; Cre-mediated recombination of the floxed allele leads to the removal of exons 6 to 11, and the formation of a PTC in exon 12; splice acceptor (SA); gray “Stop” is a PTC. (B–E) Brightfield images of 48 hpf WT (B), tnnt2a mutant (C), and tnnt2aflox/flox embryos non-injected (D) or injected at the one-cell stage with Cre mRNA (E); arrowheads and asterisks indicate respectively the presence and absence of pericardial edema. (B'–E’ ) Brightfield images and kymographs of hearts from 48 hpf WT (B’ ), tnnt2a mutant (C’ ), and tnnt2aflox/flox embryos non-injected (D’ ) or injected at the one-cell stage with Cre mRNA (E’ ); green lines outline the ventricle (V), blue lines outline the atrium (A), and vertical white lines indicate the reference axis of the kymographs. “Red hot” lookup table coloring (from Low to High) highlights the SD (B–E) or 3D variance (B’–E’ ). Diagrams indicate the anterior–posterior (A–P), dorsal–ventral (D–V), and left–right (L–R) axes.
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