Morpholino

Adult zebrafish were maintained on a 14:10 hr light:dark cycle at 28°C. Wild-type (WT; AB, Tab 14) and tp53 mutant (Berghmans et al., 2005 (link)) fish (courtesy of S. Sidi) were used. Fertilized eggs collected following natural spawning were cultured at 28°C in fish water (0.6 g/l Crystal Sea Marinemix; Marine Enterprises International, Baltimore, MD) containing methylene blue (0.002 g/l). The Mount Sinai School of Medicine Institutional Animal Care and Use Committee approved all protocols. morpholinos targeting the ATG of the mpi transcript (5’-GAGGAAACACACTTTCACTTCCGCCAT-3’), targeting the ATG of the p53 transcript (5’-GCGCCATTGCTTTGCAAGAATTG-3’), targeting the ATG of the gfpt1 transcript (5’-TCAGATACGCAAATATGCCACACAT-3’)(Senderek et al., 2011 (link)), targeting the ATG of the ogt transcript (5’-CCACGTTCCCCACCGAGCTTGCCAT-3’)(Webster et al., 2009 (link)), and a standard control morpholino (5’-CCTCTTACCTCAGTTACAATTTATA-3’) that does not target any known zebrafish transcript were obtained from Gene Tools (Philomath, OR). Needles were calibrated to inject 2 nL per embryo using a Narishige IM-300 microinjector; 0.1–4 ng of morpholino per embryo was used; 1 ng of mpi MO was identified as optimal injection amount. All injections were carried out in one- to four-cell stage embryos.

Sirt6-targeting morpholino (5′-CCCTGCTGCATAATTCACCGACATC-3′) was purchased from Gene Tools LLC (Philomath, OR, USA), and then diluted to 1uM as working concentration. For knockdown experiment, about 2.5 pl morpholino were injected into the cytoplasm of fully grown GV oocytes using a Narishige (Tokyo, Japan) microinjector. A non-targeting MO was injected as a control. In order to facilitate the morpholino-mediated knockdown of Sirt6 mRNA translation, oocytes were arrested at GV stage in M2 medium containing 2.5 μM milrinone for 20 hours, and then cultured in milrinone-free M2 medium for further experiments.