pHTBCas9 was linearized with XhoI. mRNAs of Cas9 were synthesized using Megascript T3 kit (Ambion), poly (A) tailing kit (Ambion), and Cap structure analog (New England Biolabs). pDR274 vectors encoding sgRNAs were linearized with DraI. sgRNAs were synthesized using Megascript T7 kit (Ambion). RNA was microinjected into unfertilized eggs according to a previously described method (Hikosaka et al. 1992 ). The volume of the injected media in an egg was approximately 30 pl. Electroporation of plasmids into 1-cell embryos was performed according to the previous report (Corbo et al. 1997 (link); Treen et al. 2014 (link)). Forty micrograms of the expression vector of Cas9 and 20 μg of sgRNA expression vectors were simultaneously electroporated for each electroporation. After electroporation, the embryos were washed in filtered seawater three times to remove excess plasmid DNA.
Cap structure analog
Manufactured by New England Biolabs
The Cap Structure Analog is a laboratory reagent used to mimic the cap structure found at the 5' end of eukaryotic mRNA. It serves as a tool for studying mRNA processing and translation initiation in research applications.
Automatically generated - may contain errors
4 protocols using cap structure analog
1
CRISPR/Cas9 Genome Editing in Eggs
2
Ci-pem cDNA Amplification and mRNA Synthesis
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Ci-pem cDNA was amplified by PCR and subcloned into pBS-RN328 (link) to create pRN3CipemFL. mRNA was synthesized using the Megascript T3 kit (Ambion), the poly (A) tailing kit (Ambion), and Cap structure analog (New England Biolabs). Microinjection of mRNA was performed according to a previous report29 . The concentration of mRNA in the injection medium was adjusted to 500 ngμl−1.
3
Microinjection of eGFP mRNA into Unfertilized Eggs
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eGFP mRNA was synthesized using the MEGAscript® T3 kit (Ambion, Carlsbad, CA), the Poly(A) Tailing Kit (Ambion), and Cap structure analog (New England Biolabs, Ipswich, MA) as described60 (link). We microinjected eGFP mRNA into unfertilized eggs derived from Tg[MiCiTnIGCipemG]2 or wild-type animals as described61 (link). The microinjected unfertilized eggs were fertilized by sperm of counterpart animals so as to unify the genetic background. The concentration of mRNA in the injection medium was adjusted to 500 ngμl−1. After the embryos were fixed at the appropriate stage, whole-mount in situ hybridization (WISH) was performed as described60 (link),62 (link). The eGFP fluorescence was observed with a fluorescent microscope at the late tailbud stage.
4
Transgenic Ciona Ascidian Generation
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Transgenic ascidian was generated as described previously (24 (link), 25 (link)). In brief, Minos transposase mRNA was synthesized using Megascript T3 kit, poly (A) tailing kit (Ambion, Carlsbad, CA) and Cap structure analog (New England Biolabs, Ipswich, MA). Both Minos mRNA and pMiDestF-CiVP 5’- UTR-Venus vector were electroporated into dechorionated and fertilized Ciona eggs (26 (link)). Subsequently, the electroporated eggs were cultured on agar-coated petri dishes with Millipore-filtered sea water. We raised the founder ascidian emitting Venus fluorescence, and its sperms were mated with wildtype ascidian eggs to obtain F1 progeny. Gametes of grown F1 progeny were then mated to generate transgenic F2 progeny.
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