The robust, high-throughput, and temporally regulated roxCre and loxCre reporting systems for genetic modifications in vivo

Mengyang Shi
Jie Li
Xiuxiu Liu
Kuo Liu
Wenjuan Pu
Wendong Weng
Shaohua Zhang
Huan Zhao
Kathy O. Lui
Bin Zhou author has email address

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, Shanghai, 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, Hangzhou, Chinace
Department of Chemical Pathology; and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China
School of Life Science and Technology, ShanghaiTech University, Shanghai, China

https://doi.org/10.7554/eLife.97717.1

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Figures and data

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 R26-tdT mice, R26-RSR-Cre exhibits leakiness in E13.5 embryos. Scale bars, yellow 1mm. Each image is representative of 5 individual biological samples.
(E) Examination of Cre leakiness by whole-mount fluorescence images of organs collected from R26-RSR-Cre2;R26-26-GFP adult mice. Scale bars, yellow 1mm. Each image is representative of 5 individual biological samples.
(G) Examination of Cre leakiness by whole-mount fluorescence images of organs collected from R26-R-reverseCre-R;R26-tdT adult mice. Scale bars, yellow 1mm. Each image is representative of 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. Two dual-recombinase designs for inducible Cre expression once tried before.
(J) Examination of mCre for recombination efficiency by the loxP-Stop-loxP-GFP reporter.
(K) Immunofluorescence images of cells stained with GFP on cells. Scale bars, white, 100 µm. Each image is representative of 5 individual biological samples.
(L) Quantification of the percentage of cells expressing GFP in each group. Data are the means ± SEM; n = 5.

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 R26-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 R26-Confetti allele.
(B) Fluorescence images of YFP and mCFP on liver sections collected from mice in strategy 1-3. Strategy 1 and 3 exhibit tdT or GFP in hepatocytes after DreER-rox recombination, respectively. Scale bars, 100 µm. Each image is representative of 5 individual biological samples.
(C) Quantification of the percentage of hepatocytes (Heps) expressing either YFP and/or mCFP in strategy 4-6. 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 R26-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 R26-Confetti allele.
(E) Fluorescence images of YFP and mCFP on intestinal sections collected from mice in strategy 4-6. Strategy 4 and 5 exhibit tdT or GFP in ECs after DreER-rox recombination, respectively. Scale bars, 100 µm. Each image is representative of 5 individual biological samples.
(F) Quantification of the percentage of ECs expressing either YFP and/or mCFP in strategy 4-6. Data are the means ± SEM; n = 11 mice for each strategy; ****P < 0.0001 by one-way ANOVA.

Dre-induced mCre efficiently deletes genes
(A) A schematic showing the experimental design for Dre-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 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 the day 3 post-Tam. PV, portal vein; CV, central vein. Scale bars, yellow 1mm; 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 1mm; 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 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 the 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 the tdT⁺ cells. Data are the means ± SEM; n = 6. ***P < 0.001, ****P < 0.0001 by student’s t test.

R26-loxCre-tdT is efficiently recombined by CreER
(A) A schematic showing the knock-in strategy for the generation of the R26-loxCre-tdT allele. In this line, tdT is used to denote the mCre expression after the removal of Stop.
(B) A schematic showing the experimental strategy 1 to examine the leakiness of R26-loxCre-tdT mice.
(C) A schematic showing the experimental strategy 2 to test mCre/tdT expression by AAV8-TBG-Cre.
(D) Immunostaining for tdT on tissue sections shows tdT expression in the liver and pancreas when recombination was intiated by AAV8-TBG-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;R26-tdT and Cdh5-CreER;R26-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= 6. ****P < 0.0001 by student’s t-test.

R26-loxCre-tdT enables CreER to recombine R26-Confetti efficiently
(A) Schematics showing the experimental design. In Cdh5-CreER;R26-loxCre-tdT;R26-Confetti mice, Tam-induced CreER-loxp recombination switches R26-loxCre-tdT into R26-mCre-tdT allele with simultaneous tdT labeling and expression of mCre, which subsequently targets R26-Confetti (right panel). The conventional Cdh5-CreER;R26-Confetti mice are used as control.
(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⁺ ECs in the Cdh5-CreER;R26-loxCre-tdT;R26-Confetti mice compared with that of Cdh5-CreER;R26-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.

R26-loxCre-tdT enables Alb-CreER to efficiently knockout genes in hepatocytes
(A) Schematics showing the experimental designs for Ctnnb1 gene knockout using either Alb-CreER or Alb-CreER;R26-loxCre-tdT mice.
(B) A schematic showing the experimental strategy. Alb-CreER;R26-tdT2;Ctnnb1^flox/flox and Alb-CreER;R26-loxCre-tdT;Ctnnb1^flox/+ mice were injected with Tam for 5 times, while Alb- CreER;R26-loxCre-tdT;Ctnnb1^flox/flox mice were injected with Tam for one time.
(C) Immunostaining for tdT, β-Catenin, GS, and E-CAD on liver sections. Scale bars, yellow 1mm; white 100 µm. Each image is representative of 5 individual biological samples.
(D) Immunostaining for tdT and CYP2E1 on liver sections. Scale bars, yellow 1mm; white 100 µm. Each image is representative of 5 individual biological samples.
(E) Western blotting of β-Catenin and β-Actin expression in hepatocytes, n = 3.
(F) Quantification of β-Catenin expression. Data are the means ± SEM; n = 5.
(G) qRT-PCR analysis of the relative expression of Ctnnb1 in the hepatocytes.
(H) 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, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

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