ZFIN Image: Dalgin et al., 2015, Fig. 10

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Figure Caption

Fig. 10

Neurod sgRNA/Cas9 mediated transient mutagenesis phenocopies the endocrine cell defects found in Neurod morphants. (A) Control and (B) neurod sgRNA/Cas9 injected specimens at 48 hpf. No gross morphological defects were observed. (C) T7 endonuclease I assay (T7EI) assay. (Lane 1) marker, (lane 2) untreated and (lane 3, 4) T7EI treated amplicons from control embryos. (Lane 5–15) Amplicons from embryos injected with neurod sgRNA/Cas9 were digested in varying ratios by T7EI enzyme. Asterisks indicate the PCR product that was TOPO cloned and sequenced. (D) The sequence alignment of wild type (wt), insertion (18/20, red) and deletion (2/20, green) mutations recovered from sequenced clones. Neurod sgRNA genomic target sequence (blue), protospacer adjacent motif-PAM sequence (pink). (E) Relative levels of gcga, insa, sst2, neurod and cp by real-time qPCR. Results are from 3 independent experiments and from 2 technical replicas. All values were normalized to beta-actin levels. Primer sequences are listed in Table S1.

Acknowledgments

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Reprinted from Developmental Biology, 402(1), Dalgin, G., Prince, V.E., Differential levels of Neurod establish zebrafish endocrine pancreas cell fates, 81-97, Copyright (2015) with permission from Elsevier. Full text @ Dev. Biol.