(A) Cables2 gene expression was examined by RT-PCR with ESCs, blastocyst, and E7.5 embryo samples. Gapdh was used as an internal positive control. (B–F) Wild-type embryos from E6.5 to E9.5 were …
(A) Following VelociGene’s KOMP design, the entire protein coding sequence of the target gene was deleted by homologous recombination in C57BL/6N ESCs, therefore, the full Cables2 allele was null (Ca…
(A) Genotyping by PCR analysis from whole embryo samples using three primers. Bands at 985 bp and 599 bp represent mutant and wild-type Cables2d alleles, respectively. (B) At E6.5, Cables2 was …
(A–T) All embryos were collected, genotyped, and used for WISH at E6.5. Several key gastrulation markers were examined using both wild-type and Cables2d embryos: T (n = 5), Wnt3 (n = 5), Fgf8 (n = 3)…
(A) Relative luciferase activities in 293T cells transfected with an empty control or Cables2 expression vectors together with an empty control or β-catenin expression vectors. Relative luciferase …
Co-IP showing the physical interaction of FLAG-Cables2 with endogenous β-catenin in 293T cells. Anti-GAPDH antibody was used as a negative control for evaluating specific interaction. Each …
(A–B) The EdU-incorporating cells represented the proliferation of cells in both wild-type and Cables2d/d embryos at E6.5 (n = 6). (C–F) Apoptotic cells were detected in both embryonic and …
(A) Heatmap representation of 288 genes significantly different between wild-type and Cables2d/d embryo samples (fold change >= 2, FDR < 0.05). (B) List of all downregulated (blue) and upregulated …
(A) The Cables2e1 mice was generated using CRISPR/Cas9 system. Blue marks indicate the target sites on the left and right of exon1 of Cables2. (B) RT-qPCR using adult brain samples showed the …
Expression levels of Cables2 and Rps21 in compound mutant Cables2d/e1 embryo at E9.5. Averages of three independent experiments performed in duplicated and normalized against the expression levels …
Rps21d and Rps21-indel mutant mice were generated using CRISPR/Cas9 system. One-cut target site was designed to induce the mutation in Rps21-indel. Left and right target sites were introduce (red …
(A) Schematic diagram of tetraploid complementation experiment for Cables2d Epi KO chimera. (B–E) Bright field (B, D) and YFP fluorescent (C, E) images of wild-type and Cables2d Epi KO chimeric …
Embryonic days (E) | Total number of embryos | Genotypes | ||
---|---|---|---|---|
+/+ | +/- | -/- | ||
E6.5 | 437 | 132 (30.2)* | 221 (50.6) | 80 (18.3) |
E7.5 | 70 | 18 (25.7) | 32 (45.7) | 20† (28.6) |
E8.5 | 21 | 9 (42.9) | 9 (42.9) | 3† (14.3) |
E9.5 | 18 | 7 (38.9) | 7 (38.9) | 4† (22.2) |
E12.5 | 6 | 2 (33.3) | 4 (66.7) | 0 (0) |
Adult | 90 | 24 (26.7) | 66 (73.3) | 0 (0) |
* Number of embryos (percentage), † Abnormal phenotype.
Strain | Electroporated embryos | Transfer embryos | Total number of mice | ||
---|---|---|---|---|---|
Newborn | Founder | ||||
Male | Female | ||||
Rps21d | 207 | 199 | 38 | 7 | 4 |
Rps21-indel | 274 | 200 | 0* | - | - |
* At E16.5, there were eight implanted sites.
Tetraploid embryo + Cables2d/d; ROSA26YFP/+ ESC (Epi KO chimera) | Wild-type chimera | |||||
---|---|---|---|---|---|---|
Embryonic days (E) | Total number of embryos | Phenotype | Total number of embryos | Phenotype | ||
Normal | Abnormal | Normal | Abnormal | |||
E6.5 | 2 | 1 | 1 | 4 | 3 | 1 |
E7.5 | 15 | 4 | 11 | 14 | 11 | 3 |
E8.5 | 13 | 0 | 13* | 7 | 6 | 1 |
Tetraploid embryo + Cables2d/d; ROSA26YFP/+; CAG-tdTomato-2A-Cables2-3xFLAG ESC (Epi rescue chimera) | Wild-type chimera | |||||
Embryonic days (E) | Total number of embryos | Phenotype | Total number of embryos | Phenotype | ||
Normal | Abnormal | Normal | Abnormal | |||
E7.5 | 5 | 4 | 1 | 2 | 1 | 1 |
E8.5 | 4 | 3 | 1 | 5 | 4 | 1 |
E9.5 | 2 | 2 | 0 | 3 | 2 | 1 |
* All embryos had A-P axis specification.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (M. musculus) | Cables2 | PMID:11955625 | MGI:2182335 | |
Gene (M. musculus) | Rps21 | PMID:10022917 | MGI:1913731 | |
Strain, strain background (M. musculus) | B6.Cg-Tg(TOPGAL) | Riken BRC | RBRC05918 | |
Cell line (M. musculus) | Cables2tm1(KOMP)Vlcg | KOMP | ID: VG16085, clone: 16085A-D3 RRID:MMRRC_052978-UCD | ESC line |
Cell line (Homo-sapiens) | 293T | ATCC | CRL-3216 RRID:CVCL_0063 | embryonic kidney |
Antibody | Anti-FLAG (M2) (mouse monoclonal) | Sigma-Aldrich | Cat# F1804, RRID:AB_262044 | WB(1:1000), IP (1:650) |
Antibody | Anti-β-catenin (D10A8) (rabbit monoclonal) | Cell Signaling Technology | Cat# 8480, RRID:AB_11127855 | WB(1:1000) |
Antibody | Anti-GAPDH (rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-25778, RRID:AB_10167668 | WB(1:1000) |
Antibody | Normal mouse IgG (mouse isotype control) | Santa Cruz Biotechnology | Cat# sc-2025, RRID:AB_737182 | IP(1:250) |
Antibody | Anti-Mouse IgG-HRP (secondary antibody) | GE healthcare | Cat# NA931, RRID:AB_772210 | WB(5000) |
Antibody | Anti-Rabbit IgG-HRP (secondary antibody) | GE healthcare | Cat# NA934, RRID:AB_772206 | WB(5000) |
Peptide, recombinant protein | TrueCut Cas9 Protein | Thermo Fisher Scientific | Cat# A36498 | |
Peptide, recombinant protein | Dynabeads Protein G | Thermo Fisher Scientific | Cat# 10003D | |
Commercial assay or kit | GeneArt Precision gRNA Systhesis Kit | Thermo Fisher Scientific | Cat# A29377 | |
Commercial assay or kit | Lipofectamine 3000 Reagent | Thermo Fisher Scientific | Cat# L3000015 | |
Commercial assay or kit | AmpliTag Gold 360 Master Mix | Thermo Fisher Scientific | Cat# 4398886 | |
Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat# 74104 | |
Commercial assay or kit | TB Green Premix Ex Taq II | Takara | Cat# RR820B | |
Commercial assay or kit | Dual-Glo Luciferase assay system | Promega | Cat# E2920 | |
Commercial assay or kit | Click-iT Plus EdU Imaging Kit | Thermo Fisher Scientific | Cat# C10638 | |
Commercial assay or kit | Click-iT Plus TUNEL Assay for In situ Apoptosis Detection kit | Thermo Fisher Scientific | Cat# C10619 | |
Software | CLC Genomics Workbench | Qiagen | RRID:SCR_011853 |
Differential gene expression in wild-type and Cables2d embryos.
Statistical significance was determined using ANOVA test (FDR < 0.05). This gene list is related to heatmap in Figure 6A.
Differential gene expression in wild-type and Cables2d embryos at E6.5 and KEGG pathway, GO term enrichments in upregulated genes.
Statistical significance was determined using EDGE test (FDR < 0.05). This gene list is related to Figure 6B–D.
Differential gene expression in wild-type and Cables2d embryos at E7.5 and KEGG pathway, GO term enrichments.
Statistical significance was determined using EDGE test (FDR < 0.05). This gene list is related to Figure 6E–G.
CRISPR target sites for gene-modified mice generation.
Primers for genotyping, RT-PCR and RT-qPCR.
Mendelian ratio of Cables2d and Cables2e1 intercross at E9.5.