Mo antisense oligonucleotides

All zebrafish studies were conducted with approval from the Duke University Institutional Animal Care and Use Committee. Two splice-blocking morpholino (MO) antisense oligonucleotides targeting cep55l (sb1, 5′-CTGAGATAAAGCGACTAATTCACCT-3′ and sb2, 5′-GTTGAGCTTTTCTTACCATCAGACT-3′) were obtained from Gene Tools, LLC. To determine the optimal dose for in vivo complementation assays, we injected 4, 5 and 6 ng of MO into one-to-four-cell stage zebrafish embryos obtained from natural matings of heterozygous -1.4col1a1:egfp transgenic adults.10 (link) To determine MO efficiency, we harvested embryos in Trizol (Invitrogen), extracted total RNA according to the manufacturer's instructions and generated oligo-dT-primed cDNA with the QuantiTect reverse transcription kit (Qiagen) for use as RT-PCR template. Sanger sequencing of RT-PCR products was used to identify the precise alteration of endogenous transcript. For rescue experiments, 5 ng MO and 100 pg RNA were used either independently, or combined.

The antisense MO oligonucleotides targeting individual members of the WMM complex were synthesized (Gene Tools, LLC.). The MOs (2.5-8 ng) were injected into embryos from one-cell to four-cell stage. Zebrafish WTAP and METTL3 were amplified from zebrafish cDNA library by PCR and sequentially subcloned into the pGEM-T vector and pEGFP-N1 (Clontech) to generate pWTAP-GFP or pMETTL3-GFP constructs for checking MO efficiency