Fig. 5. gRNA/Cas9-mediated gene targeting is suitable for G0 phenotyping. (A-L) Whole-mount in situ hybridization analysis of expression of the pancreas-specific marker pdip in X. tropicalis normal control tadpoles (stage 40), tadpoles injected with Cas9 mRNA (300 pg/embryo) and gRNAs (50 pg/embryo), and tadpoles injected with either ptf1a/p48GR mRNA (20 pg/embryo) alone or in combination with Cas9 and ptf1a/p48 gRNA. Dexamethasone (working concentration of 10 μM) was added at stage 14 to activate Ptf1a/p48. (A,E) Complete inhibition of pdip expression upon targeting ptf1a/p48. (B,F) Partial inhibition of pdip expression upon targeting ptf1a/p48. (C,G) Overexpression of ptf1a/p48 expands pdip expression in the territory of the stomach and duodenum. (D,H) The inhibition of pdip expression upon gRNA/Cas9-mediated targeting of ptf1a/p48 was completely rescued by co-injection of ptf1a/p48GR mRNA. (I,K) Uninjected control embryos. (J,L) As a negative control, pdip expression was unaffected upon targeting elastase. All the images are lateral views with the head to the left. E-H,K,L show further examples of the types represented in A-D,I,J, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (M,N) Dissection of 1-week-old froglets revealed severe pancreatic hypoplasia in ptf1a/p48 gRNA/Cas9-injected G0 froglets, with stomach and duodenum unaffected. The pancreas is outlined (dashed line). du, duodenum; st, stomach. (O-V) Albinism phenotype caused by tyrosinase gRNA/Cas9. (O,S) Uninjected control tadpoles. (P,T) Almost full albinism. (Q,U) Tadpoles showing severe perturbation of pigmentation. (R,V) Partial albinism. S-V show further examples of the types represented in O-R, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (W-Z) Dorsal view of adult frogs. (W) Wild type. (X) Almost full albinism caused by tyrosinase gRNA/Cas9. (Y,Z) Partial albinism. The numbers of frogs showing the illustrated phenotypes are listed. Scale bars: 400 μm in A-L; 2 mm in M,N; 1 mm on O-V; 1 cm in W-Z.
Image published in: Guo X et al. (2014)
Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
| Gene | Synonyms | Species | Stage(s) | Tissue |
|---|---|---|---|---|
| pdia2 | pdi, pdip, XPDIp | X. tropicalis | Throughout NF stage 40 | dorsal pancreatic bud ventral pancreatic bud pancreas |
Image source: Published
| Experiment + Assay | Source | Phenotypes and Disease | ||
|---|---|---|---|---|
| Xtr Wt + ptf1a CRISPR + NF40 (in situ hybridization) | Fig. 5 AE |
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| Xtr Wt + ptf1a CRISPR + juvenile (whole-mount microscopy) | Fig. 5 N |
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| Xtr Wt + tyr CRISPR + NF40 (whole-mount microscopy) | Fig. 5 PQRTUV |
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| Xtr Wt + tyr CRISPR + adult (whole-mount microscopy) | Fig. 5 XYZ |
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