Ecorv

Different restriction enzymes were selected to digest the RCA products according to the species’ mtDNA reference sequence to generate two distinguishable bands in 0.5% agarose gel. For mouse, EcoRV (NEB) was used to digest RCA products into two long fragments (9.5 kb and 6.8 kb). For human, SacI (NEB) was used to digest RCA products into two long fragments (9.6 kb and 6.9 kb). For fruit fly, NdeI (NEB) and EcoRV (NEB) were used to digest RCA products into two long fragments (10 kb and 9.8 kb). For plasmid pTEsindbisGFP, EcoRV (NEB) was used to digest RCA products into two long fragments (10.4 kb and 4.1 kb). All digestion reactions were carried out according to the manufacturer’s protocol.

The coding sequences for CT26-5 and MC38-7 transgenes were purchased as phosphorylated gBlock dsDNA fragments (IDT) and cloned into p-tetOCMV-BGHpA, containg CMV promoter with two TetO repeats and a BGH polyA, previously digested with EcoRV (New England Biolabs). The CDS for CT26-31 was generated by Gibson assembly (New England Biolabs) of two overlapping gBlock sequences (Integrated DNA Technologies) into p-tetOCMV-BGHpA previously digested with EcoRV and Not1 restriction enzymes (New England Biolabs). The expression cassettes were then transferred into pGAd plasmid, containing the E1/E3/E4 deleted in which the E4 is replaced with Ad5 E4 ORF6 of a Great Ape Adenovirus (serotype group C). The transgene cassette was introduced into the E1 deletion by homologous recombination in BJ5183 cells (Agilent). GAds vectors were then produced by transfection of adherent M9 cells (293 cells derivative) with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) and amplification in suspension M9 cells. Vectors were then purified from infected cells by Vivapure Adenopack 20 RT (Sartorius).

pExpress-1 vectors containing complete stub1 cDNA sequence (ENSDART00000145075.3, ensemble genome database; Ensembl release 102—November 2020) was purchased (Source BioScience, Nottingham, United Kingdom) and verified by Sanger sequencing. Sense- and anti-sense cRNA probes were synthesized for 687 bp zebrafish stub1 sequence (C70-C755) following plasmid linearization with NotI (New England BioLabs) and EcoRV (New England BioLabs) restriction enzymes, respectively, and purification of PCR-amplified stub1 cDNA fragments containing SP6 (NotI-digested) and T7 (EcoRV-digested) promoter sites. PCR reactions were prepared by using forward and reverse primers for T7- (forward: 5′-CTGTTCCTCAGCCGCAAGTA-3′, reverse: 5′-TAATACGACTCACTATAGGGGTCAAAATGGCCAACACGC T-3′) and SP6- (forward: 5′-ATTTAGGTGACACTATAGAA CTGTTCCTCAGCCGCAAGTA-3′, reverse: 5′-GTCAAAAT GGCCAACACGCT-3′) containing fragments. Probe synthesis was performed using Digoxigenin (DIG) RNA labeling kit (Roche Molecular Biochemicals, Basel, Switzerland) and 400 ng of PCR product as template. Transcription reactions were terminated by addition of EDTA on ice, and cRNA probes were precipitated in a solution of 0.1 × volume of 4 M LiCl, 3.7 μg/μl tRNA (Roche), and 3 × volume of 100% ethanol.