Viable endogenous eGFP-tagging recapitulates the Tnnt2a expression pattern. (A) Schematics of tnnt2a locus showing the insertion of an eGFP cassette at the C-terminus of the protein using the CRISPR/Cas9 knock-in vector pGTag; the knock-in cassette contains the last exon of tnnt2a and an eGFP before the stop codon, separated by a GSSS linker, and it is inserted in the last intron; 48 bp homology arms (HA) flanking the Cas9 cleavage site were used and the vector is cleaved at a universal (U) CRISPR site; the tnnt2abns511 allele contains a concatemer composed of multiple insert and vector backbone copies, which was removed by injecting Cre mRNA at the one-cell stage to create the tnnt2abns513 allele. (B–D) Brightfield images of 48 hpf tnnt2amn0031Gt/+ (B), tnnt2amn0031Gt/bns511 (C), and tnnt2amn0031Gt/bns513 (D) embryos; arrowhead and asterisks indicate, respectively, the presence and absence of pericardial edema. (B'–D') Brightfield images and kymographs of hearts from 48 hpf tnnt2amn0031Gt/+ (B’ ), tnnt2amn0031Gt/bns511 (C’ ), and tnnt2amn0031Gt/bns513 (D’ ) embryos; green lines outline the ventricle (V), blue lines outline the atrium (A), and vertical white lines indicate the reference axis of the kymographs. (E) Confocal image of a heart from a 48 hpf tnnt2abns513/bns513 embryo stained for F-actin with Phalloidin; maximum z-projection; annotations correspond to the ventricle (V) and atrium (A). “Red hot” lookup table coloring (from Low to High) highlights the SD (B–D) or 3D variance (B’–D’ ). Diagrams indicate the anterior–posterior (A–P), dorsal–ventral (D–V), and left–right (L–R) axes.
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