Two antisense morpholinos (MOs; Gene Tools, Philomath, OR), 1 targeting the translational ATG site (tnnt1 TR-MO) and 1 targeting the splice acceptor site between intron 7 and exon 8 (tnnt1 SP8-MO), were designed to knock down the zebrafish tnnt1 gene (Ensembl-ID: ENSDARG00000037954). The MO sequences were tnnt1 TR-MO: 5’-ATTCCTCTTCTACATCA GACATGAT-3’ and tnnt1 SP8-MO: 5’-CCTGTAATGTGGAG GACAAAAGCAA-3’. MOs were dissolved in ×1 Danieau buffer with 0.1% phenol red. Five nanograms of tnnt1 TR-MO and 1ng of tnnt1SP8-MO were injected into the fertilized yolks of 1-cell embryos.
For rescue experiments, full-length Homo sapiens TNNT1 (NM_003283.6) cDNA was cloned into a pCS2+ vector (from Dr Nathan D. Lawson, Addgene plasmid #22423) using Gateway technology (Invitrogen, Carlsbad, CA). The TNNT1 c.287T>C (p.Leu96Pro) substitution was introduced using the Q5 Site-Directed Mutagenesis Kit (Invitrogen). The mRNAs for both wild-type (WT) and c.287T>C mutant constructs were synthesized in vitro using mMessenger Kit (Ambion, Austin, TX) and purified using mini Quick Spin RNA columns (Roche, Basel, Switzerland). One hundred picograms of mRNA was injected into randomly selected 1-cell stage embryos.
For rescue experiments, full-length Homo sapiens TNNT1 (NM_003283.6) cDNA was cloned into a pCS2+ vector (from Dr Nathan D. Lawson, Addgene plasmid #22423) using Gateway technology (Invitrogen, Carlsbad, CA). The TNNT1 c.287T>C (p.Leu96Pro) substitution was introduced using the Q5 Site-Directed Mutagenesis Kit (Invitrogen). The mRNAs for both wild-type (WT) and c.287T>C mutant constructs were synthesized in vitro using mMessenger Kit (Ambion, Austin, TX) and purified using mini Quick Spin RNA columns (Roche, Basel, Switzerland). One hundred picograms of mRNA was injected into randomly selected 1-cell stage embryos.