Mmessage mmachin kit

The nexmifa splice-blocking Morpholino (MO) and the standard control MO (Std MO) were synthesized by Gene Tools. The sequences are: 5′-AAAATGGTAGGAGTTATAAATGAGT-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3, respectively. MOs were diluted to 0.3 mM in RNase-free water, injected into one-cell stage embryos, and then raised in E3 medium at 28.5°C to generate nexmifa knockdown embryos (morphants). To perform rescue experiments, we generated nexmifa mRNA, efna5b mRNA, and sema6ba mRNA in vitro. Briefly, we cloned zebrafish nexmifa, efna5b, and sema6ba separately into PCS2⁺ vectors. Next, we linearized plasmids, then in vitro synthesized mRNA using the mMESSAGE mMACHIN Kit (Ambion, Austin, Texas, United States) according to manufacturer’s instructions. Finally, we purified Capped mRNAs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). MOs or mRNAs were injected into the yolk of one cell stage embryos using borosilicate glass capillaries (Sarasota, Florida, United States) and a PV830 pneumatic picopump (Sarasota, Florida, United States).

Translation blocking antisense Morpholino (MOs; Gene Tools) against the ATG-containing sequence was designed (5′-AAATCCTCTGGGCATCTTCGCCAGC-3′) to target the translation start site according to the manufacturer's instruction and the other MO oligo (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as standard control. The MOs were diluted to 0.3 mM with RNase-free water and injected into the yolk of one to two-cell stage embryos and then raised in E3 medium at 28.5°C.
The cDNAs containing the open reading frame of the target genes were cloned into PCS2⁺ vector respectively and then were transcribed in vitro using the mMESSAGE mMACHIN Kit (Ambion, USA) after the recombinant plasmids linearized with NotI Restriction Enzyme (NEB, England), and then the capped mRNAs were purified by RNeasy Mini Kit (Qiagen, Germany). 2 nl target genes and mCherry mRNA mixture (1:1) were injected at 20 ng/μl into 1/2-cell stage embryos.

The claudin h mRNA was transcribed in vitro using linearized artificial PCS2+ vector with the claudin h open reading frame cDNAs by the mMESSAGE mMACHIN Kit according to the manufacturer’s instruction (Ambion, United States). After purified using RNeasy Mini Kit (Qiagen, Germany), 2 nl capped mRNA was co-injected with claudin h Mo into one-cell stage embryos.

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.