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In this work, a transgenic zebrafish line that expresses the fluorophore dsRed under the endogenous zebrafish cochlin promotor is supposed to be established, using the CRISPR/Cas9 system. dsRed was cloned into a pBluescript vector, followed by the cloning of the cochlin locus into this vector. This bait construct was then supposed to be micro injected into wild type AB zebrafish embryos. The micro injection of Cas9 mRNA, single guide RNA and a bait construct was practiced with the tyrosinase gene, which was disrupted using CRISPR/Cas9.
Vicia faba leaves and calli were transformed using CRISPR Cas RNP. Two kinds of CPP fused SpyCas9 were used with sgRNA7, sgRNA5 or sgRNA13 targeting PDS exon 1, PDS exon 2 or MgCh exon 3 respectively. RNP were applied using high pressure spraying, biolistic delivery, incubation in RNP solution and infiltration of leaf tissue. A PCR and restriction enzyme based approach was used for detection of mutation. Screening of 679 E. coli colonies containing the cloned fragments resulted in detection of 14 mutations. Most of the 14 mutations were deletions of sizes 150, 500 or 730 bp. 5 out of the 14 mutations were point mutations located two to three bp upstream of PAM.