Nebasechanger online tool

We used site-directed mutagenesis to generate point mutations/substitutions in human MTTP complementary DNA cloned in pcDNA3 plasmid as described earlier (8 (link), 15 (link), 38 (link)). In brief, the forward and reverse primers (Table S2) for each mutation were designed using NEBase changer online tool from New England Biolab (NEB) website as per their protocol. The Q5 site-directed mutagenesis kit (catalog no.: E0554; NEB) was used for PCR amplification of pcDNA3-MTTP-FLAG. The PCR amplification was confirmed by resolving the PCR product on 0.8% agarose gel. Then, 1 μl (50–100 ng) of the PCR product was subjected to KLD enzyme reaction as suggested in the kit, and the product was transformed into Escherichia coli–competent cells supplied in the kit. The transformants were selected on LB-agar plates containing 100 μg/ml of carbenicillin antibiotic. Plasmid was isolated from antibiotic-resistant E. coli colonies, and DNA sequencing (Genewiz) was performed to confirm the presence of desired mutation. Colonies with the desired mutations were grown in 200 ml LB broth, and the plasmid was isolated using ZymoPURII plasmid maxi prep kit (catalog no.: D4204; Zymo Research).

The full-length mpv17 and mpv17l2 cDNAs from wild-type strain and human MPV17 cDNA from pCR2.1 TOPO vector, kindly provided by the Zeviani laboratory (University of Cambridge, Cambridge, UK), were subcloned into the pCS2+ vector. The human MPV17 p.R50Q (CGG>CAG) mutated form was generated, starting from wild-type construct, using a Q5^® Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward, 5′-CAGAGAGGCCAGACTCTGACCATG-3′; reverse, 5′-GTGTTCCTGCAGACCCCG-3′, both designed by NEBaseChanger online tool (New England Biolabs). Capped mRNA was transcribed using the mMessage mMachine^® kit (Thermo Fisher Scientific) according to the manufacturer's protocol. The mRNA was resuspended in water and microinjected into one-cell mpv17^−/− embryos at 70 ng/μl.

pCDNA3 plasmids harboring ALK2^WT, ALK3^WT and ALK6^WT were purchased from Addgene (#80870, #80873 and #80882). Point mutations in ALK2, ALK3 and ALK6 were made in-house using the Q5-Site Directed Mutagenesis Kit from New England Biolabs. Mutagenic oligonucleotides were designed using the NEBaseChanger online tool (New England Biolabs) and purchased from Integrated DNA Technologies. Mutations were confirmed by automated Sanger sequencing (Genewiz).