For mRNA synthesis or cell transfection, the corresponding cDNA sequence was inserted into mCherry-tagged pXT7, Flag-tagged pCS2, Myc-tagged pCMV5 or GST-tagged PEBG1 vector. dnrock2a was used as previously described (Zhang et al., 2009 (link)). All mRNAs were synthesized using the mMessage mMachine T7/SP6 kit (Ambion).
Mmessage mmachine sp6 t7 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MMESSAGE mMACHINE SP6/T7 kit is a laboratory product designed for in vitro transcription. It provides reagents and protocols to synthesize capped and polyadenylated messenger RNA (mRNA) using the SP6 or T7 RNA polymerase.
Automatically generated - may contain errors
5 protocols using mmessage mmachine sp6 t7 kit
1
mRNA Synthesis and Cell Transfection
2
Morpholino-mediated Knockdown of Zebrafish Signaling Genes
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Morpholinos of gnrh3 antisense MO1 (5′-cactccatgctaaaactgctgtgtt-3′), MO2 (5′-ggaccagcaaccttcctttccactc-3′), MO3 (5′-gcaaccttcctttccactccatgct-3′), control MO1 (5′-aacacagcagttttagcatggagtg-3′), csnk1a (5′-ccatgtcctaaaatccgagaagtac-3′), gsk3b (5′- gagtaaaatacgtctgtttgtcttg-3′), control MO2 (5′-cctcttacctcagttacaatttata-3′) and fgfr1 (5′-gcagcagcgtggtcttcattatcat-3′) (GeneTools, Corvallis, Oregon) were diluted to 10 ng/nl and injected into the yolk of 1-cell embryos at 3–15 ng/injection. Capped RNA was synthesized with mMESSAGE mMACHINE T7/SP6 kit (Ambion, Austin, Taxas) from linearized plasmids. Full-length gnrh3 (100 pg) [1] (link), gnrh2 (100 pg) [1] (link), constitutively active PKA catalytic subunit PKA* (50 pg) [28] (link), PKI (dominant-negative regulatory subunit of protein kinase A, 100 pg) [28] (link), shha (2 ng) [29] (link) and shhb (twhh, 2 ng) [29] (link) mRNA was injected into the yolk of 1-cell embryos, and embryos were allowed to develop at 28.5°C.
3
CRISPR Targeting of aqp8ab Exon 3
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The sgRNA targeting the third exon of
aqp8ab was designed by using the CRISPR online tool (
https://zlab.bio/guide-design-resources ). Effective targeting site is: 5′-GGTGACTCTGGTGGTCCTGA-3′. The mRNA of
aqp8ab sgRNA and
Cas9 were synthesized
in vitro using the MAXISCRIPT T7 kit (Ambion, Austin, USA) and mMESSAGE mMACHINE SP6/T7 kit (Ambion), respectively. Then the mRNA was purified using the RNeasy Mini kit (Qiagen, Hilden, Germany), and dissolved in RNase free Ultapure water (Thermo Fisher Scientific, Waltham, USA).
aqp8ab was designed by using the CRISPR online tool (
aqp8ab sgRNA and
Cas9 were synthesized
in vitro using the MAXISCRIPT T7 kit (Ambion, Austin, USA) and mMESSAGE mMACHINE SP6/T7 kit (Ambion), respectively. Then the mRNA was purified using the RNeasy Mini kit (Qiagen, Hilden, Germany), and dissolved in RNase free Ultapure water (Thermo Fisher Scientific, Waltham, USA).
4
Zebrafish arl4aa Gene Knockdown
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Antisense MO (Gene Tools, Philomath, OR) was designed to target the translation start codon of zebrafish arl4aa. A random sequence MO was used as negative control. To test the molecular specificity of MO, 5′ UTR of arl4aa gene containing the MO binding sequence was cloned into the BamHI-EcoRI site of the pEGFP-N3 vector (Clontech, Palo Alto, CA) and the plasmid (25 pg) was injected into one-cell-stage embryos with or without MO. To test if mRNA can rescue the hematopoietic defects induced by MO, arl4aa complete coding sequence was cloned to pGEMT easy vector (Promega, Madison, WI) and capped mRNA was synthesized using the mMESSAGE mMACHINE SP6/T7 Kit (Ambion, Austin, TX). Silent mutations with 4-bp mismatch were introduced into the MO binding sequence using arl4aa-rescue F primer (Table S1 ) to generate an arl4aa mRNA that was resistant to MO. MO (1.0 ng) and mRNA (50 pg) were injected into 1- to 4-cell stage embryos.
5
Synthesis of Chimeric mRNAs with Vasa 3'UTRs
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Chimeric mRNAs which fused EGFP or mCherry to the 3′UTR of Sebastes schlegelii or Danio rerio vasa gene were synthesized by in vitro transcription, and the protocols followed the methods described previously (Zhou et al., 2019 (link)). The main process is as follows: firstly, template plasmids were constructed using Gibson assembly, and the 3′UTRs of EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR plasmids in our lab were replaced by vasa 3′UTRs of black rockfish and zebrafish, respectively. Then, according to the plasmids, the transcription templates were synthesized by PCR. After purification, capped chimeric mRNAs were synthesized using mMESSAGE mMACHINE SP6/T7 kit (Ambion, United States). The primers for preparing mRNAs are also shown in Table 1 . In the previous study, the EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR mRNAs that contained red seabream and marine medaka vasa 3′UTRs have been synthesized in our lab (Zhou et al., 2019 (link)).
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