Generation of a self-cleaved inducible Cre recombinase for efficient temporal genetic manipulation

Abstract

Site-specific recombinase-mediated genetic technology, such as inducible Cre-loxP recombination (CreER), is widely used for in vivo genetic manipulation with temporal control. The Cre-loxP technology improves our understanding on the in vivo function of specific genes in organ development, tissue regeneration, and disease progression. However, inducible CreER often remains inefficient in gene deletion. In order to improve the efficiency of gene manipulation, we generated a self-cleaved inducible CreER (sCreER) that switches inducible CreER into a constitutively active Cre by itself. We generated endocardial driver Npr3-sCreER and fibroblast driver Col1a2-sCreER, and compared them with conventional Npr3-CreER and Col1a2-CreER, respectively. For easy-to-recombine alleles such as R26-tdTomato, there was no significant difference in recombination efficiency between sCreER and the conventional CreER. However, for alleles that were relatively inert for recombination such as R26-Confetti, R26-LZLT, R26-GFP, or VEGFR2^flox/flox alleles, sCreER showed a significantly higher efficiency in recombination compared with conventional CreER in endocardial cells or fibroblasts. Compared with conventional CreER, sCreER significantly enhances the efficiency of recombination to induce gene expression or gene deletion, allowing temporal yet effective in vivo genomic modification for studying gene function in specific cell lineages.

Keywords: Cre-loxP; cell lineages; gene deletion; genetic manipulation.

Figure 1. Generation and characterization of sCreER

1. Schematic diagram showing the working principle for sCreER.

2. Schematic diagram showing steps of switch of sCreER into Cre.

3. Generation of Npr3‐sCreER or Npr3‐CreER knock‐in alleles by homologous recombination.

4. Schematic diagram showing the experimental strategy.

5. Whole‐mount fluorescence view of E14.5‐15.5 hearts from Npr3‐sCreER;R26‐tdTomato and Npr3‐CreER;R26‐tdTomato mice.

6. Immunostaining for tdTomato, ESR, and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato⁺ESR⁻ endocardial cells.

7. Quantification of the percentage of tdTomato⁺ or ESR⁺ endothelial cells. Data are mean ± SEM; n = 5.

8. Isolation of tdTomato⁻ or tdTomato⁺ cells from E14.5 Npr3‐sCreER;R26‐tdTomato and Npr3‐CreER;R26‐tdTomato mice by FACS.

9. qRT–PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato⁺ cells. Data are mean ± SEM; n = 5; *P < 0.05.

10. Western blotting of ESR and GAPDH in tdTomato⁻ and tdTomato⁺ cells. Data are mean ± SEM; n = 5; *P < 0.05; n.s., non‐significant.

Data information: Scale bars, 500 μm. Each figure is representative of 5 individual biological samples.

Figure 2. sCreER has significantly higher recombination efficiency than CreER by R26‐Confetti reporter

1. A

   Schematic diagram showing recombination readouts by crossing of Npr3‐CreER or Npr3‐sCreER with R26‐Confetti reporter.

2. B

   Schematic figure showing experimental strategy.

3. C

   Whole‐mount fluorescence images of E14.5 Npr3‐CreER;R26‐Confetti and Npr3‐sCreER;R26‐Confetti mouse hearts. No Tam is used as control for leakiness of Npr3‐sCreER.

4. D, E

   Immunostaining for CDH5 on heart sections shows a significant increase in endothelial cell labeling in Npr3‐sCreER;R26‐Confetti heart compared with Npr3‐CreER;R26‐Confetti heart (Tam). *P < 0.05; data are mean ± SEM; n = 5.

Data information: Scale bars, 500 μm in (C); 100 μm in (D). Each image is representative of 5 individual biological samples.

Figure 3. Recombination efficiency by sCreER or CreER on R26‐LZLT reporter

1. Schematic diagram showing Npr3‐sCreER or Npr3‐CreER mouse crossed with a newly generated Rosa26 reporter R26‐LZLT.

2. Schematic diagram showing experimental strategy.

3. Whole‐mount fluorescent images of E14.5 or E15.5 Npr3‐sCreER;R26‐LZLT and Npr3‐CreER;R26‐LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER.

4. Representative immunostaining images for tdTomato, ESR, and CDH5 on heart sections.

5. Quantification of the percentage of endocardial cells expressing tdTomato in Npr3‐sCreER;R26‐LZLT (sCreER) or Npr3‐CreER;R26‐LZLT (CreER) hearts. Data are mean ± SEM; n = 5; P?value was determined using the unpaired Student's t‐test (*P < 0.05).

Data information: Scale bars, 500 μm. Each figure is a representative of 5 individual biological samples.

Figure 4. Increased efficiency of gene deletion by sCreER, compared with CreER

1. A

   Schematic diagram showing Kdr gene deletion by sCreER.

2. B

   Schematic figure showing experimental design.

3. C

   Immunostaining for VEGFR2 on Npr3‐sCreER;Kdr ^flox/flox heart section from mice without tamoxifen treatment (No Tam).

4. D, E

   Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.

5. F, G

   Immunostaining for tdTomato, VEGFR2, and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2⁺ endocardial cells (ECs) in tdTomato⁺ (tdT⁺) cells in the TM regions. Cartoon images (bottom) indicate tdTomato⁺ cells (red) and VEGFR2⁺ cells (green) in the TM or compact myocardium (CM). Data are mean ± SEM; n = 5.

Data information: Scale bars, 100 μm. Each image is a representative of 5 individual biological samples.

Figure 5. Generation and characterization of Col1a2‐sCreER and Col1a2‐CreER alleles

1. Schematic diagram showing knock‐in of sCreER or CreER into Col1a2 locus by homologous recombination.

2. Schematic figure showing experimental strategy.

3. Whole‐mount fluorescence images of Col1a2‐sCreER;R26‐tdTomato or Col1a2‐CreER;R26‐tdTomato hearts. Inserts indicate bright‐field images.

4. Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals.

5. Immunostaining for tdTomato and ESR on heart sections. Arrowheads indicate valve mesenchyme.

6. Quantification of the percentage of PDGFRa⁺ cells expressing tdTomato in Col1a2‐sCreER;R26‐tdTomato (sCreER) or Col1a2‐CreER;R26‐tdTomato (CreER) hearts. n.s., non‐significant; data are mean ± SEM; n = 6; n.s., non‐significant.

7. Quantification of the percentage of tdTomato⁺ cells expressing ESR. *P < 0.05; data are mean ± SEM; n = 6; *P < 0.05.

8. Immunostaining for PDGFRa in heart sections shows a significant increase in fibroblast labeling in valves of Col1a2‐sCreER;R26‐Confetti heart compared with Col1a2‐CreER;R26‐Confetti heart (Tam).

9. Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± SEM; n = 5; *P < 0.05.

Data information: P?value was determined using the unpaired Student's t‐test in (F, G, I). Scale bars, 500 μm in (C–E); 100 μm in (H). Each figure is a representative of 5–6 individual biological samples.