The robust, high-throughput, and temporally regulated roxCre and loxCre reporting systems for genetic modifications in vivo
- CAS CEMCS-CUHK Joint Laboratories, New Cornerstone Science Laboratory, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China
- Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, China
- School of Life Sciences, Westlake University, China
- Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, China
- School of Life Science and Technology, ShanghaiTech University, China
Figures
Figure 1
Design of roxCre for DreER-induced mCre expression.
(A) A schematic showing genetic labeling and/or gene knockout in CreER-expressing cells. (B, D, F) Strategies for DreER-induced Cre expression. (C) Crossing with Rosa26-tdT mice, Rosa26-RSR-Cre exhibits leakiness in E13.5 embryos. Scale bars, yellow 1 mm. Each image represents 5 individual biological samples. (E) Examination of Cre leakiness by whole-mount fluorescence images of organs collected from Rosa26-RSR-Cre2;Rosa26-GFP adult mice. Scale bars, yellow 1 mm. Each image represents 5 individual biological samples. (G) Examination of Cre leakiness by whole-mount fluorescence images of organs collected from Rosa26-R-reverseCre-R;Rosa26-tdT adult mice. Scale bars, yellow 1 mm. Each image represents 5 individual biological samples. (H) A schematic showing the design for roxCre and mCre. (I) A schematic showing mCre1 to mCre12 by insertion of rox into Cre cDNA at 12 different loci. (J) Examination of mCre for recombination efficiency by the loxP-Stop-loxP-GFP reporter. (K) Immunofluorescence images of cells stained with GFP and DAPI. Scale bars, white, 100 µm. Each image represents 5 individual biological samples. (L) Quantification of the percentage of cells expressing GFP in each group. Data are the means ± SEM; n=5.
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Figure 1—source data 1
Numerical data to generate Figure 1L.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig1-data1-v1.xlsx
Figure 2 with 5 supplements
DreER-induced mCre robustly recombines inert alleles.
(A) A schematic showing the experimental design to test the recombination efficiency of mCre1 and mCre7 on the Rosa26-Confetti allele. Tam-induced DreER-rox recombination leads to mCre1/tdT or mCre7/GFP expression in hepatocytes in Strategy 1 or 2, respectively. Strategy 3 uses conventional Alb-CreER as control. YFP and mCFP signals are used for the examination of recombination on the Rosa26-Confetti allele. (B) Fluorescence images of YFP and mCFP on liver sections collected from mice in Strategies 1–3. Strategies 1 and 2 exhibit tdT or GFP in hepatocytes after DreER-rox recombination, respectively. Scale bars, 100 µm. Each image is representative of 11 individual biological samples. (C) Quantification of the percentage of hepatocytes (Heps) expressing either YFP and/or mCFP in Strategies 1–3. Data are the means ± SEM; n=11 mice for each strategy; ****p<0.0001 by one-way ANOVA. (D) A schematic showing the experimental strategy to test the recombination efficiency of mCre4 and mCre10 on Rosa26-Confetti allele. Tam-induced recombination leads to mCre4/tdT or mCre10/GFP expression in endothelial cells (ECs) in Strategy 4 or 5, respectively. Strategy 6 uses conventional Cdh5-CreER as control. YFP and mCFP signals are used for the examination of recombination on Rosa26-Confetti allele. (E) Fluorescence images of YFP and mCFP on intestinal sections collected from mice in Strategies 4–6. Strategies 4 and 5 exhibit tdT or GFP in ECs after DreER-rox recombination, respectively. Scale bars, 100 µm. Each image is representative of 11 individual biological samples. (F) Quantification of the percentage of ECs expressing either YFP and/or mCFP in Strategies 4–6. Data are the means ± SEM; n=11 mice for each strategy; ****p<0.0001 by one-way ANOVA.
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Figure 2—source data 1
Numerical data to generate Figure 2.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
No leakiness in Alb-roxCre1-tdT;Rosa26-GFP and Cdh5-roxCre4-tdT;Rosa26-GFP mice.
(A) Schematics showing the mating strategy for examining the leakiness of roxCre1 and fluorescence reporters. (B) Schematics showing the experimental strategy of Alb-roxCre1-tdT;Rosa26-GFP. (C) Whole-mount fluorescence and immunostaining sections of Alb-roxCre1-tdT;Rosa26-GFP mice. Inserts are bright-field images. Scale bars, yellow, 1 mm; white, 100 µm. Each image represents 5 individual biological samples. (D) Schematics showing the mating strategy for examining the leakiness of roxCre4 and fluorescence reporters. (E) Schematics showing the experimental strategy of Cdh5-roxCre4-tdT;Rosa26-GFP. (F) Whole-mount fluorescence and immunostaining sections of Cdh5-roxCre4-tdT;Rosa26-GFP mice. Inserts are bright-field images. Scale bars, yellow, 1 mm; white, 100 µm. Each image represents 5 individual biological samples.
Figure 2—figure supplement 2
No leakiness in Alb-roxCre7-GFP;Rosa26-tdT and Cdh5-roxCre10-GFP;Rosa26-tdT mice.
(A) Schematics showing the mating strategy for examining the leakiness of roxCre7 and fluorescence reporters. (B) Schematics showing the experimental strategy of Alb-roxCre7-GFP;Rosa26-tdT. (C) Whole-mount fluorescence and immunostaining sections of Alb-roxCre7-GFP;Rosa26-tdT mice. Inserts are bright-field images. Scale bars, yellow, 1 mm; white, 100 µm. Each image represents 5 individual biological samples. (D) Schematics showing the mating strategy for examining the leakiness of roxCre10 and fluorescence reporters. (E) Schematics showing the experimental strategy of Cdh5-roxCre10-GFP;Rosa26-tdT. (F) Whole-mount fluorescence and immunostaining sections of Cdh5-roxCre10-GFP;Rosa26-tdT mice. Inserts are bright-field images. Scale bars, yellow, 1 mm; white, 100 µm. Each image represents 5 individual biological samples.
Figure 2—figure supplement 3
Limitations of readily recombined reporters in assessing mCre-loxP recombination efficiency.
(A) The design of the mating strategy. (B) The experimental strategy. (C) Gating strategy for quantifying the proportion of tdT+ and GFP+ hepatocytes in Rosa26-DreER;Alb-roxCre1-tdT;Rosa26-GFP mice, under untreated and tamoxifen-injected conditions. (D) Gating strategy for quantifying the proportion of GFP+ and tdT+ hepatocytes in Rosa26-DreER;Alb-roxCre7-GFP;Rosa26-tdT mice, under untreated and tamoxifen-injected conditions. (E) The dot plots demonstrate that the ratio of Rosa26-reporter+ hepatocytes within mCre1-tdT+ hepatocytes is comparable to that within mCre7-GFP+ hepatocytes. (F) Immunostaining for GFP, tdT, and DAPI in liver sections from Rosa26-DreER;Alb-roxCre1-tdT;Rosa26-GFP and Rosa26-DreER;Alb-roxCre7-GFP;Rosa26-tdT mice without tamoxifen injection. (G) Immunostaining for GFP and tdT in liver sections from tamoxifen-induced Rosa26-DreER;Alb-roxCre1-tdT;Rosa26-GFP and Rosa26-DreER;Alb-roxCre7-GFP;Rosa26-tdT mice. Scale bars, 100 μm. Images are representative of 5 independent biological replicates.
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Figure 2—figure supplement 3—source data 1
Numerical data to generate Figure 2—figure supplement 3E.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig2-figsupp3-data1-v1.xlsx
Figure 2—figure supplement 4
Cre-loxP recombination efficiency among individual mice.
(A) CreER mouse lines with limited recombination efficiency can recombine the Rosa26-Confetti reporter to express YFP, nuclear GFP (nGFP), membrane CFP (mCFP), or RFP. (B) Tamoxifen-induced Dre-rox recombination generates mCre mice with enhanced recombination efficiency, which mediates persistent inversion of the sequence between two inversely oriented loxP sites. (C) Immunostaining for GFP (anti-GFP antibody also recognizes YFP and CFP), RFP, and DAPI in tissue sections from Rosa26-DreER;Alb-roxCre1-tdT;Rosa26-Confetti, Rosa26-DreER;Cdh5-roxCre4-tdT;Rosa26-Confetti, Rosa26-DreER;Alb-roxCre7-GFP;Rosa26-Confetti, and Rosa26-DreER;Cdh5-roxCre10-GFP;Rosa26-Confetti mice without tamoxifen injection. Scale bars, 100 μm. Images are representative of 5 independent biological replicates.
Figure 2—figure supplement 5
Comparison of recombination efficiency mediated by mCre4 and mCre10.
(A) YFP and CFP fluorescence in tissue sections from Rosa26-DreER;Cdh5-roxCre4-tdT;Rosa26-Confetti and Rosa26-DreER;Cdh5-roxCre10-GFP;Rosa26-Confetti mice. (B) Quantification of the percentage of endothelial cells (ECs) expressing YFP or CFP. Data are the means ± SEM; n=5. *p<0.05, ***p<0.001, ****p<0.0001 by Student’s t-test. Scale bars, 100 μm. Images are representative of 5 independent biological replicates.
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Figure 2—figure supplement 5—source data 1
Numerical data to generate Figure 2—figure supplement 5B.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig2-figsupp5-data1-v1.xlsx
Figure 3 with 1 supplement
DreER-induced mCre efficiently deletes genes in specific cell subpopulations.
(A) A schematic showing the experimental design for DreER-induced mCre expression and the subsequent gene deletion. (B) A schematic showing the experimental strategy. (C and D) qRT-PCR analysis of the relative expression of Ctnnb1 (C) and Glul (D) in the sorted tdT+ hepatocytes. Data are the means ± SEM; n=5; ****p<0.0001 by Student’s t-test. (E) A schematic showing the experimental strategy. (F) Immunostaining for tdT, β-catenin, GS, and E-CAD on liver sections collected on day 3 post-Tam. PV, portal vein; CV, central vein. Scale bars, yellow 1 mm; white 100 µm. Each image is representative of 5 individual biological samples. (G) Immunostaining for tdT, β-catenin, GS, and E-CAD on liver sections collected at week 4 post-Tam. Scale bars, yellow 1 mm; white 100 µm. Each image is representative of 5 individual biological samples. (H) A schematic showing the experimental strategy. (I) Western blotting of β-catenin and GAPDH in sorted tdT+ cells. (J) Quantification of the relative expression of β-catenin protein. Data are the means ± SEM; n=3. **p<0.01 by Student’s t-test. (K) qRT-PCR analysis of the relative expression of Ctnnb1 in sorted tdT+ cells. Data are the means ± SEM; n=6. ****p<0.0001 by Student’s t-test. (L) qRT-PCR analysis of the relative expression of Glul, Axin2, Cyp1a2, Cyp2e1, Oat, Tcf7, Lect2, Tbx3, Slc1a2, and Rhbg in sorted tdT+ cells. Data are the means ± SEM; n=6. ***p<0.001, ****p<0.0001 by Student’s t-test.
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Figure 3—source data 1
Numerical data to generate Figure 3.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig3-data1-v1.xlsx
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Figure 3—source data 2
Original files for western blot analysis are displayed in Figure 3I.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig3-data2-v1.zip
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Figure 3—source data 3
PDF files containing original western blot for Figure 3I, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig3-data3-v1.zip
Figure 3—figure supplement 1
Generation and characterization of the Cyp2e1-DreER mouse line.
(A) Schematic of the CRISPR/Cas9-mediated knock-in strategy for generating the Cyp2e1-DreER mouse line. (B) The genetic lineage tracing strategy. (C) Whole-mount fluorescence images of livers from Cyp2e1-DreER;Rosa26-RSR-tdT mice with or without tamoxifen administration. Insets show corresponding bright-field images. (D) Immunostaining for tdT, CK19, and HNF4a on liver sections. Yellow arrowheads, tdT+HNF4a+ hepatocytes. (E) Immunostaining for tdT, DAPI, and the periportal hepatocyte marker E-cadherin (E-CAD) reveals that most tdT+ hepatocytes reside in the pericentral zone rather than the periportal region. (F) Immunostaining for tdT and the epithelial cell marker EpCAM shows tdT+ cells are not biliary epithelial cells. (G) Whole-mount fluorescence imaging and immunofluorescent staining of kidney sections from Cyp2e1-DreER;Rosa26-RSR-tdT (left panel) and Cyp2e1-DreER;Alb-roxCre-tdT (right panel) mice. Scale bars: yellow, 1 mm; white, 100 µm. Images are representative of 5 independent biological replicates.
Figure 4 with 1 supplement
Rosa26-loxCre-tdT is efficiently recombined by CreER.
(A) A schematic showing the knock-in strategy for the generation of the Rosa26-loxCre-tdT allele. In this line, tdT is used to denote the mCre expression after the removal of Stop. (B) A schematic showing experimental Strategy 1 to examine the leakiness of Rosa26-loxCre-tdT mice. (C) A schematic showing experimental Strategy 2 to test mCre/tdT expression by AAV2/8-hTBG-Cre. (D) Immunostaining for tdT on tissue sections shows tdT expression in the liver and pancreas when recombination was initiated by AAV2/8-hTBG-Cre. Scale bars, white 100 µm. Each image is representative of 5 individual biological samples. (E) A schematic showing the experimental Strategies 3 and 4 for comparing the CreER-mediated first recombination efficiency between Cdh5-CreER;Rosa26-tdT and Cdh5-CreER;Rosa26-loxCre-tdT mice. (F) Immunostaining for tdT and VE-Cad on liver sections collected on day 7 post-Tam. Scale bars, white 100 µm. Each image is representative of 5 individual biological samples. (G) Quantification of the percentage of VE-Cad+ ECs expressing tdT. Data are the means ± SEM; n=5. ****p<0.0001 by Student’s t-test.
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Figure 4—source data 1
Numerical data to generate Figure 4G.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Characterization of the Rosa26-loxCre-tdT mouse line.
(A) No leakiness was observed for the Rosa26-loxCre-tdT mouse line following intercrossing with Rosa26-tdT2 mice. (B) The Rosa26-loxCre-tdT mouse line effectively attenuates the leakiness observed in certain CreER lines, such as Cdh5-CreER. Data are presented as means ± SEM; n=5 mice. p<0.01 by Student’s t-test. Scale bars: white, 100 µm. Each image represents 5 independent biological replicates.
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Figure 4—figure supplement 1—source data 1
Numerical data to generate Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig4-figsupp1-data1-v1.xlsx
Figure 5
Rosa26-loxCre-tdT enables CreER to recombine Rosa26-Confetti efficiently.
(A) Schematics showing the experimental design. In Cdh5-CreER;Rosa26-loxCre-tdT;Rosa26-Confetti mice, Tam-induced CreER-loxP recombination switches Rosa26-loxCre-tdT into Rosa26-mCre-tdT allele with simultaneous tdT labeling and expression of mCre, which subsequently targets Rosa26-Confetti (right panel). The conventional Cdh5-CreER;Rosa26-Confetti mice are used as controls. (B) A schematic showing the experimental strategy. (C) Whole-mount YFP and mCFP fluorescence images of retina collected from two mice groups. Scale bars, white 100 µm. Each image is representative of 5 individual biological samples. (D) Immunofluorescence images of YFP, mCFP, and VE-Cad on tissue sections show significantly more YFP+ and/or mCFP+ endothelial cells (ECs) in the Cdh5-CreER;Rosa26-loxCre-tdT;Rosa26-Confetti mice compared with those of Cdh5-CreER;Rosa26-Confetti mice (left panel). The right panel shows the quantification of ECs expressing YFP and/or mCFP. Data are the means ± SEM; n=5. ****p<0.0001 by Student’s t-test. Scale bars, white 100 µm. Each image is representative of 5 individual biological samples.
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Figure 5—source data 1
Numerical data to generate Figure 5D.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig5-data1-v1.xlsx
Figure 6
Rosa26-loxCre-tdT adaptor ensures efficient recombination.
(A) The working strategies for Alb-CreER;Rosa26-tdT2;Rosa26-Confetti, Alb-CreER;iSuRe-Cre;Rosa26-Confetti, and Alb-CreER;Rosa26-loxCre-tdT;Rosa26-Confetti. (B) The experimental strategy. (C) Fluorescence images of YFP, mCFP, and tdT on the liver sections. The red arrows point out some tdT–YFP+ or tdT–mCFP+ hepatocytes. The white arrows point out some tdT+YFP+ or tdT+mCFP+ hepatocytes. The cyan arrows point out some tdT+YFP– or tdT+CFP– hepatocytes. (D) Quantification of the tdT+ hepatocytes. (E) Quantification of the percentage of tdT+ hepatocytes expressing YFP and/or mCFP. Data are means ± SEM; n=6. ****p<0.0001 by one-way ANOVA. Scale bars, white 100 µm. Each image is representative of 6 individual biological samples.
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Figure 6—source data 1
Numerical data to generate Figure 6.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig6-data1-v1.xlsx
Figure 7 with 2 supplements
Rosa26-loxCre-tdT enables Alb-CreER to efficiently knock out genes in hepatocytes.
(A) Schematics showing the experimental designs for Ctnnb1 gene knockout using either Alb-CreER;Rosa26-tdT2 or Alb-CreER;Rosa26-loxCre-tdT mice. (B) A schematic showing the experimental strategy. Alb-CreER;Rosa26-tdT2;Ctnnb1flox/flox and Alb-CreER;Rosa26-loxCre-tdT;Ctnnb1flox/+ mice were injected with Tam for five times, while Alb-CreER;Rosa26-loxCre-tdT;Ctnnb1flox/flox mice were injected with Tam once. (C) Immunostaining for tdT, β-catenin, GS, and E-CAD on liver sections. Scale bars, yellow 1 mm; white 100 µm. Each image is representative of 5 individual biological samples. (D) Western blotting of β-catenin and β-actin expression in hepatocytes, n=3. (E) Quantification of β-catenin expression. Data are the means ± SEM; n=5. (F) qRT-PCR analysis of the relative expression of Ctnnb1 in the hepatocytes. (G) qRT-PCR analysis of the relative expression of Glul, Axin2, Cyp2e1, Oat, Tcf7, Lect2, Tbx3, and Slc1a2 in hepatocytes. Data are the means ± SEM; n=7; ns, nonsignificant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA.
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Figure 7—source data 1
Numerical data to generate Figure 7.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-data1-v1.xlsx
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Figure 7—source data 2
Original files for western blot analysis are displayed in Figure 7D.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-data2-v1.zip
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Figure 7—source data 3
PDF files containing original western blot for Figure 7D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-data3-v1.zip
Figure 7—figure supplement 1
Ctnnb1 was specifically knocked out in tdT+ hepatocytes in the Alb-CreER;Rosa26-loxCre-tdT mouse line.
(A) The Alb-CreER;Rosa26-loxCre-tdT mouse line exhibited no leaky fluorescent signal. (B) Fluorescence-activated cell sorting (FACS) sorting of tdT+ hepatocytes from Alb-CreER;Rosa26-tdT;Ctnnb1flox/flox mice at 4 weeks after a single tamoxifen administration. (C) Relative Ctnnb1 mRNA expression analyzed by qRT-PCR. Data are presented as means ± SEM; n=6. ns, not significant as determined by Student’s t-test. (D) Immunostaining for tdT, GS, E-CAD, and DAPI on liver sections of Alb-CreER;Rosa26-tdT;Ctnnb1flox/flox. (E) tdT+ hepatocytes and tdT– cells were sorted from Alb-CreER;Rosa26-loxCre-tdT;Ctnnb1flox/flox mice at 3 weeks after a concentration-gradient tamoxifen administration, and subjected to qRT-PCR analysis of relative Ctnnb1 expression. Data are presented as means ± SEM; n=5. ****p<0.0001 by Student’s t-test. (F) Dose-response curves showing the ratio of tdT+ hepatocytes by FACS (top), GS+ hepatocytes by immunostaining (middle), and E-CAD+ hepatocytes by immunostaining (bottom), plotted against log5-transformed tamoxifen concentrations. (G) Immunostaining for tdT, GS, E-CAD, and DAPI on liver sections of Alb-CreER;Rosa26-tdT;Ctnnb1flox/flox. Scale bars: white, 100 µm. Images are representative of 5 independent biological replicates.
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Figure 7—figure supplement 1—source data 1
Numerical data to generate Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-figsupp1-data1-v1.xlsx
Figure 7—figure supplement 2
Genotyping information for the newly established mouse lines in this study.
Mouse line construction strategy, primer design, representative PCR genotyping outcomes, and full primer sequences corresponding to the six new mouse lines characterized in this study.
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Figure 7—figure supplement 2—source data 1
The original files for the agarose gel electrophoresis results are shown in Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-figsupp2-data1-v1.zip
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Figure 7—figure supplement 2—source data 2
PDF files containing the agarose gel electrophoresis results of Figure 7—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97717/elife-97717-fig7-figsupp2-data2-v1.zip
Author response image 1
Leakiness in Alb CreER;iSuRe-Cre mouse line.
Pictures are representative results for 5 mice. Scale bars, white 100 µm.
Author response image 2
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Rosa26-DreER | Li et al., 2018 | 10.1161/CIRCULATIONAHA.118.034250 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Alb-CreER | He et al., 2017 | 10.1038/nm.4437 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Rosa26-GFP | Zhang et al., 2016a | 10.1161/CIRCRESAHA.116.308749 | Generated in a previous study |
| strain, strain background (Mus musculus) | Rosa26-tdT | Madisen et al., 2010 | 10.1038/nn.2467 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Rosa26-tdT2 | Liu et al., 2020 | 10.1074/jbc.RA119.011349 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Rosa26-RSR-tdT | Zhang et al., 2016a | 10.1161/CIRCRESAHA.115.307202 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Rosa26-Confetti | Snippert et al., 2010 | 10.1016/j.cell.2010.09.016 | Generated in a previous study |
| Strain, strain background (Mus musculus) | iSuRe-Cre | Fernández-Chacón et al., 2019 | 10.1038/s41467-019-10239-4 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Ctnnb1-flox | Huelsken et al., 2001 | doi: 10.1016/s0092-8674(01)00336-1 | Generated in a previous study |
| Strain, strain background (Mus musculus) | Rosa26-RSR-Cre | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Rosa26-RSR-Cre2 | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Rosa26-R-reverseCre-R | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Cdh5-CreER | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Alb-roxCre1-tdT | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Alb-roxCre7-GFP | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Cdh5-roxCre4-tdT | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Cdh5-rox10-GFP | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Cyp2e1-DreER | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Strain, strain background (Mus musculus) | Rosa26-loxCre-tdT | This paper | This paper | Generated in this study and available from the corresponding author upon request |
| Transfected construct (Mus musculus) | AAV2/8-TBG-Cre | Taitool: Cat# S0657-8-H5 | AAV2/8 vector expressing Cre under the TBG promoter | |
| Recombinant DNA reagent | pcDNA3.1 (plasmid) | Invitrogen: Cat# V79020 | Parental plasmid from which mutants were generated | |
| Recombinant DNA reagent | pCAG-loxp-stop-loxp-ZsGreen (plasmid) | Addgene: Cat# 51269 | Reporter plasmid used to monitor Cre activity | |
| Recombinant DNA reagent | pHR-CMV-nlsCRE (plasmid) | Invitrogen: Cat# 12265 | Vector used to express Cre recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre1 (plasmid) | This paper | Vector used to express mCre1 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre2 (plasmid) | This paper | Vector used to express mCre2 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre3 (plasmid) | This paper | Vector used to express mCre3 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre4 (plasmid) | This paper | Vector used to express mCre4 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre5 (plasmid) | This paper | Vector used to express mCre5 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre6 (plasmid) | This paper | Vector used to express mCre6 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre7 (plasmid) | This paper | Vector used to express mCre7 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre8 (plasmid) | This paper | Vector used to express mCre8 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre9 (plasmid) | This paper | Vector used to express mCre9 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre10 (plasmid) | This paper | Vector used to express mCre10 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre11 (plasmid) | This paper | Vector used to express mCre11 recombinase | |
| Recombinant DNA reagent | pcDNA3.1-mCre12 (plasmid) | This paper | Vector used to express mCre12 recombinase | |
| Cell line (Homo sapiens) | HEK293A | ZQXZbio: Cat# ZQ0941 | RRID:CVCL_6910 | |
| Antibody | Donkey Polyclonal anti-mouse Fab antibody | Jackson: Cat# 715-007-003 | RRID:AB_2307338 | (20 µg/mL). Used for minimizing primary antibody cross-linking and avoiding Fc-mediated artifacts |
| Antibody | Rabbit Polyclonal anti-RFP | Rockland: Cat# 600-401-379 | RRID:AB_2209751 | IF (1:1000) |
| Antibody | Goat Polyclonal anti-RFP | Rockland: Cat# 200-101-379 | RRID:AB_2744552 | IF (1:1000) |
| Antibody | Rabbit Polyclonal anti-GFP | Invitrogen: Cat# A11122 | RRID:AB_221569 | IF (1:500) |
| Antibody | Goat Polyclonal anti-GFP | Rockland: Cat# 600-101-215M | RRID:AB_2612804 | IF (1:500) |
| Antibody | Rat monoclonal anti-GFP | Nacalai: Cat# 04404-84 | RRID:AB_2313654 | IF (1:500) |
| Antibody | Rabbit Polyclonal anti-Glutamine Synthetase | Abcam: Cat# ab49873 | RRID:AB_880241 | IF (1:10,000) |
| Antibody | Mouse monoclonal anti-β-catenin | BD Pharmingen: Cat# 610153 | RRID:AB_397555 | IF (1:200) WB (1:5000) |
| Antibody | Goat Polyclonal anti-E-cadherin | R&D: Cat# AF748 | RRID:AB_355568 | IF (1:500) |
| Antibody | Rat monoclonal anti-EpCAM | Abcam: Cat# ab92382 | RRID:AB_2049615 | IF (1:2000) |
| Antibody | Goat Polyclonal anti-VE-cadherin | R&D: Cat# AF1002 | RRID:AB_2077789 | IF (1:100) |
| Antibody | Rabbit Polyclonal anti-CK19 | AbboMax: Cat# 602-670 | RRID:AB_3720929 | IF (1:500) |
| Antibody | Rabbit monoclonal HNF4α (C11F12) Rabbit mA | Cell Signaling Technology: Cat# 3113 | RRID:AB_2295208 | IF (1:1000) |
| Antibody | Mouse monoclonal anti-GAPDH | Proteintech: Cat# 60004-1-IG | RRID:AB_2107436 | WB (1:5000) |
| Antibody | Rabbit Polyclonal anti-β-actin | Epizyme: Cat# LF202 | RRID:AB_3094632 | WB (1:5000) |
| Antibody | Donkey Polyclonal HRP-conjugated Donkey anti-mouse IgG | JIR: Cat# 715-035-150 | RRID:AB_2340770 | WB (1:5000) |
| Antibody | Goat Polyclonal HRP-conjugated Goat anti-rabbit IgG | JIR: Cat# 111-035-047 | RRID:AB_2337940 | WB (1:5000) |
| Chemical compound, drug | Tamoxifen | Sigma | Cat# T5648 | |
| Chemical compound, drug | Paraformaldehyde (PFA) | Sigma | Cat# P6148-500g | |
| Chemical compound, drug | Triton X-100 | Sigma | Cat# T9284 | |
| Chemical compound, drug | 4'6-Diamidino-2-phenylindole (DAPI) | Vector Lab | Cat# D21490 | |
| Chemical compound, drug | Collagenase type I | Gibco | Cat# 17100-017 | |
| Chemical compound, drug | Percoll | GE Healthcare | Cat# 17-0891-01 | |
| Chemical compound, drug | DNase I | Worthington | Cat# LS002139 | |
| Chemical compound, drug | TRIzol | Invitrogen | Cat# 15596018 | |
| Chemical compound, drug | RIPA lysis buffer | Beyotime | Cat# P0013B | |
| Chemical compound, drug | Protease inhibitors | Roche | Cat# 11836153001 | |
| Chemical compound, drug | 5× loading buffer | Beyotime | Cat# P0015L | |
| Chemical compound, drug | Precast gradient gels | Beyotime | Cat# P0469M | |
| Chemical compound, drug | Immobilon PVDF membranes | Millipore | Cat# IPVH00010 | |
| Chemical compound, drug | 1× running buffer | Epizyme | Cat# PS105S | |
| Chemical compound, drug | Blocking buffer | Epizyme | Cat# PS108P | |
| Chemical compound, drug | Primary antibody dilution buffer | Epizyme | Cat# PS114 | |
| Chemical compound, drug | 1× TBST | Epizyme | Cat# PS103S | |
| Commercial assay or kit | Lipofectamine 3000 Transfection Reagent | Thermo Fisher | Cat# L3000015 | |
| Commercial assay or kit | Prime Script RT kit | Takara | Cat# RR047A | |
| Commercial assay or kit | SYBR Green qPCR master mix | Thermo Fisher | Cat# 4367659 | |
| Commercial assay or kit | Pierce BCA Protein Assay kits | Thermo Scientific | Cat# 23227 | |
| Commercial assay or kit | Chemiluminescent HRP substrate | Thermo Fisher | Cat# WBKLS0500 | |
| Software, algorithm | Fiji | Version: 2.9.0/1.54f | RRID:SCR_002285 | https://imagej.net/software/fiji/ |
| Software, algorithm | GraphPad Prism | Version: 9.5.1 | RRID:SCR_002798 | https://www.graphpad.com |
| Software, algorithm | FlowJo | Version: 10.9.0 | RRID:SCR_008520 | https://www.flowjo.com |
| Software, algorithm | Photoline | Version: 23.02 | RRID:SCR_027878 | http://pl64.com |
| Other | Olympus microscope BX53 | Olympus | Used for image acquisition after immunofluorescence staining | |
| Other | Olympus confocal FV3000 | Olympus | Used for image acquisition after immunofluorescence staining | |
| Other | Zeiss stereoscopic microscope AxioZoom V16 | Zeiss | Used for whole-mount image acquisition | |
| Other | Nikon A1 FLIM | Nikon | Used for image acquisition after immunofluorescence staining | |
| Other | Leica SP8 WILL | Leica | Used for image acquisition after immunofluorescence staining | |
| Other | QuantStudio 6 Real-Time PCR System | Thermo Fisher | Used for the quantitative detection of qPCR | |
| Other | MiniChemi 610 Plus | Biogamma | Used for acquiring western blot results | |
| Other | Beckman Coulter CytoFLEX LX 5-Laser Stem Cell Multicolor Flow Cytometer | Beckman | Used for flow cytometry analysis | |
| Other | FACSAria SORP | Becton, Dickinson and Company | Used for flow cytometry sorting | |
| Other | Sony MA900 | Sony | Used for flow cytometry sorting |
Additional files
-
Supplementary file 1
Mouse genotypes.
The subheadings in this document correspond to individual figures. The leftmost column indicates the lettered labels within each figure. The middle column provides descriptive annotations for the experimental groups. The right column specifies the detailed mouse genotypes for each respective group.
- https://cdn.elifesciences.org/articles/97717/elife-97717-supp1-v1.docx
-
Supplementary file 2
Oligos for M. musculus genes.
The leftmost column indicates the names of genes. The middle column provides related sequences. The right column describes the additional information on primers.
- https://cdn.elifesciences.org/articles/97717/elife-97717-supp2-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/97717/elife-97717-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The robust, high-throughput, and temporally regulated roxCre and loxCre reporting systems for genetic modifications in vivo
eLife 13:RP97717.
https://doi.org/10.7554/eLife.97717.4
