Lifetechmultiste gateway manual

Full-length and truncated coding sequences of sema6D were synthesized and inserted into PCS²⁺ vector as templates for in vitro transcription. The mRNA synthesis was carried out by using the mMESSAGEmMACHINESp6 Ultra Kit (Ambion) and purified with the MEGAclearTM Transcription Clean-Up Kit (Ambion) before the injection. Finally, 2 nl capped mRNA was co-injected with sema6D Mo into one-cell stage embryos. The fli1a:sema6D plasmid was constructed by LR recombination as described in the LifetechMultiste Gateway Manual (Life Technologies, Carls-bad, CA, USA). Then, the construct was injected into one cell stage embryos of Tg(mnx1:EGFP::kdrl:ras-mcherry) zebrafish for tissue specific rescue experiments (1 ng per embryo).

Translation-blocking morpholino (5′-GCTCGGTTTCCATCATGTTATACAT-3′) against the ATG-containing sequence was synthesized by Gene Tools. Morpholino was diluted to 0.3 mM with RNase-free water and injected into one-cell stage embryos and then raised in E3 medium at 28.5°C for imaging.
The Sox2 cDNAs containing the open reading frame were cloned into PCS2⁺ vector. After this recombinant plasmid was linearized, the Sox2 mRNA was synthesized in vitro by the mMESSAGE mMACHIN Kit (Ambion, USA) according to the manufacturer’s instruction, and then the capped mRNAs were purified using RNeasy Mini Kit (Qiagen, Hilden, Germany). Two nanoliters of Sox2 mRNA was injected at 20 ng/μl into one-cell stage embryos.
The Sox2 ORF was cloned from the zebrafish by PCR, and then cloned into a pME-MCS vector to produce middle entry clone (pME-Sox2). p5E-mnx1 plasmid was obtained from Addgene. To generate an expression construct, p5E-mnx1, pME-Sox2, and p3E-polyA were combined with pDestTol2pA2 by the LR recombination reaction as described in the Lifetech Multiste Gateway Manual (Figure 5E; Life Technologies, Carlsbad, CA, USA). Subsequently, this construct was co-injected with tol2-transposase mRNA into one-cell stage Sox2 mutant zebrafish embryos to create the mosaic rescue zebrafish model.