Method of constructing zebrafish notch1a mutants
A method of constructing a zebrafish notch1a mutant using CRISPR/Cas9 technique. The method includes: determining a target for knocking out notch1a; using primers T7-notch1a-sfd and tracr rev for PCR amplification with a pUC19-gRNA scaffold plasmid as a template; transcribing PCR product in vitro followed by purification to obtain gRNA; and microinjecting the gRNA and a Cas9 mRNA into a zebrafish embryo followed by culture to obtain an notch1a mutant of stable inheritance. The invention selects a specific target and utilizes CRISPR/Cas9 technique to knock out the notch1a in the zebrafish without destroying other genes, generating the zebrafish notch1a mutant. Moreover, the invention also discloses the phenotype of the zebrafish notch1a mutant, which plays a significant role in studying the effect of the Notch1a receptor in the Notch signaling pathway.
Shanghai Ocean University
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Qinghua Ce
Zhang Ji