Generation of a self‐cleaved inducible Cre recombinase for efficient temporal genetic manipulation
2020; Springer Nature; Volume: 39; Issue: 4 Linguagem: Inglês
10.15252/embj.2019102675
ISSN1460-2075
AutoresXueying Tian, Lingjuan He, Kuo Liu, Wenjuan Pu, Huan Zhao, Yan Li, Xiuxiu Liu, Muxue Tang, Ruilin Sun, Jian Fei, Yong Ji, Zengyong Qiao, Kathy O. Lui, Bin Zhou,
Tópico(s)Pluripotent Stem Cells Research
ResumoResource14 January 2020free access Transparent process Generation of a self-cleaved inducible Cre recombinase for efficient temporal genetic manipulation Xueying Tian Corresponding Author [email protected] Key Laboratory of Regenerative Medicine of Ministry of Education, College of Life Science and Technology, Jinan University, Guangzhou, China The 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 Search for more papers by this author Lingjuan He The 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 Search for more papers by this author Kuo Liu The 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 School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Wenjuan Pu The 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 Search for more papers by this author Huan Zhao The 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 Search for more papers by this author Yan Li The 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 Search for more papers by this author Xiuxiu Liu The 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 Search for more papers by this author Muxue Tang The 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 Search for more papers by this author Ruilin Sun Shanghai Model Organisms Center, Inc., Shanghai, China Search for more papers by this author Jian Fei Shanghai Model Organisms Center, Inc., Shanghai, China Search for more papers by this author Yong Ji The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Zengyong Qiao Department of Cardiovascular Medicine, Southern Medical University Affiliated Fengxian Hospital, Shanghai, China Search for more papers by this author Kathy O Lui orcid.org/0000-0002-1616-3643 Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Sha Tin, Hong Kong SAR, China Search for more papers by this author Bin Zhou Corresponding Author [email protected] orcid.org/0000-0001-5278-5522 Key Laboratory of Regenerative Medicine of Ministry of Education, College of Life Science and Technology, Jinan University, Guangzhou, China The 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 School of Life Science and Technology, Shanghai Tech University, Shanghai, China The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Xueying Tian Corresponding Author [email protected] Key Laboratory of Regenerative Medicine of Ministry of Education, College of Life Science and Technology, Jinan University, Guangzhou, China The 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 Search for more papers by this author Lingjuan He The 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 Search for more papers by this author Kuo Liu The 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 School of Life Science and Technology, Shanghai Tech University, Shanghai, China Search for more papers by this author Wenjuan Pu The 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 Search for more papers by this author Huan Zhao The 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 Search for more papers by this author Yan Li The 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 Search for more papers by this author Xiuxiu Liu The 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 Search for more papers by this author Muxue Tang The 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 Search for more papers by this author Ruilin Sun Shanghai Model Organisms Center, Inc., Shanghai, China Search for more papers by this author Jian Fei Shanghai Model Organisms Center, Inc., Shanghai, China Search for more papers by this author Yong Ji The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Zengyong Qiao Department of Cardiovascular Medicine, Southern Medical University Affiliated Fengxian Hospital, Shanghai, China Search for more papers by this author Kathy O Lui orcid.org/0000-0002-1616-3643 Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Sha Tin, Hong Kong SAR, China Search for more papers by this author Bin Zhou Corresponding Author [email protected] orcid.org/0000-0001-5278-5522 Key Laboratory of Regenerative Medicine of Ministry of Education, College of Life Science and Technology, Jinan University, Guangzhou, China The 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 School of Life Science and Technology, Shanghai Tech University, Shanghai, China The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China Search for more papers by this author Author Information Xueying Tian *,1,2, Lingjuan He2, Kuo Liu2,3, Wenjuan Pu2, Huan Zhao2, Yan Li2, Xiuxiu Liu2, Muxue Tang2, Ruilin Sun4, Jian Fei4, Yong Ji5,6, Zengyong Qiao7, Kathy O Lui8 and Bin Zhou *,1,2,3,5 1Key Laboratory of Regenerative Medicine of Ministry of Education, College of Life Science and Technology, Jinan University, Guangzhou, China 2The 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 3School of Life Science and Technology, Shanghai Tech University, Shanghai, China 4Shanghai Model Organisms Center, Inc., Shanghai, China 5The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China 6Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China 7Department of Cardiovascular Medicine, Southern Medical University Affiliated Fengxian Hospital, Shanghai, China 8Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Sha Tin, Hong Kong SAR, China *Corresponding author. Tel: +86 20 85222687; E-mail: [email protected] *Corresponding author. Tel: +86 21 54920974; E-mail: [email protected] EMBO J (2020)39:e102675https://doi.org/10.15252/embj.2019102675 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 VEGFR2flox/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. Synopsis A self-cleaved CreER (sCreER) is generated by placing two loxP sites on the flanks of ER cassette. After tamoxifen (Tam) induction, sCreER protein is transported into the nucleus where it cleaves loxP flanked ER, converting sCreER DNA into Cre genotype. Thus, the Cre recombinase can be switched from an inducible form to a constitutively active form. sCreER significantly enhances the efficiency of gene manipulation on some alleles that are relatively inert for recombination. sCreER exhibits temporal control property by tamoxifen. sCreER self-cleaves and converts to Cre for robust gene deletion. sCreER is more efficient than CreER in recombination of a refractory or less susceptible target locus. Introduction Site-specific recombinase (SSR), such as Cre (Sauer & Henderson, 1988; Nagy, 2000), has been widely used in genetic manipulations such as lineage tracing, ectopic gene expression, or tissue-specific gene deletion. This genetic recombination system provides a critical basis for understanding in vivo cell behavior and gene function in organ development, tissue regeneration, and disease progression (Buckingham & Meilhac, 2011; Kretzschmar & Watt, 2012). The most widely used inducible SSR system is CreER, a fusion protein that contains both Cre recombinase and mutated hormone-binding domain of the estrogen receptor, ER (Feil et al, 1997; Vermot et al, 2003). While Cre recombinase can move freely into the nucleus for DNA recombination, CreER is retained in the cytoplasm and remains inactive for DNA recombination until synthetic estrogen receptor ligand, such as tamoxifen, binds to CreER, which then allows it to transport from cytoplasm into nucleus for subsequent excision of floxed genomic DNA (Tian et al, 2015). Compared with the constitutively active Cre, inducible CreER allows external control of its activity, enhancing the precision of genetic manipulation (e.g., gene deletion) in a temporal manner (Zhang et al, 2016a, 2018). Despite its advantage in allowing temporal control and being extensively used in many laboratories, conventional CreER driven by some promoters is inefficient in gene deletion. In many conditions, multiple times of tamoxifen treatment, sometimes over 10 times, have to be used for gene deletion (Melendez et al, 2009; Sorensen et al, 2009; Parkhurst et al, 2013; Zhou et al, 2014; Shankman et al, 2015; Van Keymeulen et al, 2015), which would have side effect on animal health in long term. Here, we aim to generate a new system that could switch CreER into a constitutively active Cre for enhancing efficiency while retaining the temporal control property. Results Generation and characterization of the Npr3-sCreER allele We generated a self-cleaved CreER (sCreER), in which the ER cassette was flanked by two loxP sites (Fig 1A). After tamoxifen (Tam) induction, sCreER protein was transported into the nucleus and cleaved loxP-flanked ER, leaving only Cre DNA driven by a specific gene promoter (Fig 1B). Thus, the Cre recombinase can be switched from an inducible form to a constitutively active form (Fig 1B). To directly test the technical feasibility of sCreER, we generated two knock-in mouse lines, namely Npr3-sCreER and Npr3-CreER, in which the sCreER or CreER cDNA was targeted into the same endogenous translation start codon ATG of the endogenous Npr3 gene (Fig 1C). We first compared the recombination efficiency of Npr3-sCreER and Npr3-CreER by crossing them with R26-tdTomato (Madisen et al, 2010), which is a very sensitive reporter for Cre-loxP recombination. Immunostaining of ESR (estrogen receptor) for detection of sCreER or CreER protein on E11.5 and E12.5 Npr3-sCreER or Npr3-CreER heart sections, respectively, showed its expression in endocardial cells of the trabecular myocardium (TM, Appendix Fig S1), in consistent with the reported Npr3 expression pattern (Zhang et al, 2016a). We then treated mice with Tam at E11.5 and collected hearts for analysis at E13.5–E15.5 (Fig 1D). Whole-mount fluorescence images showed robust tdTomato signals inside the atria and ventricles of Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato hearts after Tam treatment, respectively (Fig 1E; Appendix Fig S2A). However, negligible tdTomato signal was observed in these hearts without Tam treatment (Fig 1E). Immunostaining for tdTomato and ESR on heart sections showed that the majority of endocardial cells located in the TM were tdTomato+ in both Npr3-sCreER and Npr3-CreER hearts (Fig 1F and G; Appendix Fig S2B), indicating that Npr3-sCreER worked as efficiently and specifically as conventional Npr3-CreER in recombination of the R26-tdTomato allele. Noticeably, while ESR expression could be readily detected in the Npr3-CreER heart treated with Tam, we observed significant reduction in ESR+ cells in the Npr3-sCreER heart treated with Tam (arrowheads, Fig 1F and G). In Npr3-sCreER heart samples without Tam, sCreER expression (examined by ESR immunostaining) could still be readily detected (Fig 1F and G). Figure 1. Generation and characterization of sCreER Schematic diagram showing the working principle for sCreER. Schematic diagram showing steps of switch of sCreER into Cre. Generation of Npr3-sCreER or Npr3-CreER knock-in alleles by homologous recombination. Schematic diagram showing the experimental strategy. Whole-mount fluorescence view of E14.5-15.5 hearts from Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice. Immunostaining for tdTomato, ESR, and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato+ESR− endocardial cells. Quantification of the percentage of tdTomato+ or ESR+ endothelial cells. Data are mean ± SEM; n = 5. Isolation of tdTomato− or tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice by FACS. qRT–PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato+ cells. Data are mean ± SEM; n = 5; *P < 0.05. 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. Download figure Download PowerPoint Additionally, qRT–PCR analysis showed a significant reduction in ER mRNA expression in tdTomato+ cells isolated by fluorescence-activated cell sorting (FACS) from Npr3-sCreER compared with that of Npr3-CreER (Fig 1H). As a control, the respective expression levels of Cre, Npr3, and Cdh5 mRNA remained comparable between Npr3-sCreER and Npr3-CreER labeled cells (Fig 1I). Western blot of isolated tdTomato− and tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato or Npr3-CreER;R26-tdTomato showed a significant reduction in ESR expression level of tdTomato+ cells isolated from Npr3-sCreER;R26-tdTomato mice (Fig 1J). Taken together, this proof-of-principle experiment demonstrated that sCreER could work as efficiently as CreER to label through a sensitive reporter such as R26-tdTomato, and, at the DNA level, sCreER genotype could be switched to Cre genotype after self-cleavage of ER (Fig 1A and B). To further test the sCreER system at different developmental stages and in adults, we compared conventional CreER and sCreER driver lines for recombination efficiency after single low-dose Tam administration at late embryonic stage (E16.5) and the adult stages (30 weeks). Based on the R26-tdTomato reporter, we found that single low-dose Tam led to similar efficiency in endocardial cell labeling of the Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice when low-dose Tam was induced at a late embryonic stage (Appendix Fig S3). Notably, Npr3-sCreER worked more efficiently than Npr3-CreER when single low dose of Tam was administered at the adult stage (Appendix Fig S4). To assess the leakiness of sCreER in the absence of Tam, we collected Npr3-sCreER;R26-tdTomato hearts at the adult stage of 30 weeks old and found that most endocardial cells in the ventricles were negative for tdTomato, while a subset of endocardial cells in the atria expressed tdTomato (Appendix Fig S5). sCreER efficiently recombines allele that is inert for recombination by conventional CreER We next asked whether sCreER has advantage over CreER in targeting genomic DNA regions that were relatively inert for recombination. We first took advantage of the R26-Confetti allele, which is known to be difficult for recombination, and thus has been widely used in sparse cell clonal analysis (Snippert et al, 2010; Ritsma et al, 2014). Crossing Npr3-CreER or Npr3-sCreER with R26-Confetti allele yields four distinct fluorescent colors in each of the four cells, respectively (Fig 2A). We adopted the same Tam induction strategy as in R26-tdTomato reporter analysis (Fig 2B). Whole-mount views of hearts showed sparse fluorescent signal in Npr3-CreER;R26-Confetti hearts, while a remarkably intensive fluorescent signal was detected in Npr3-sCreER;R26-Confetti hearts (Fig 2C). As a control, there was no detectable fluorescent signal in Npr3-sCreER;R26-Confetti hearts without Tam (Fig 2C), indicating that strong sCreER for R26-Confetti reporter recombination is controllable. Since the constitutively active Cre switched from sCreER would flip R26-confetti reporters continuously, we could detect some overlapping fluorescence signals (Fig 2C). We interpreted that the fluorescence proteins before being flipped over had not fully degraded when new reporter gene started expression in the same cell. Immunostaining on heart sections showed a > 30-fold increase in the percentage of endocardial cells labeled in Npr3-sCreER compared with that of Npr3-CreER (51.82 ± 4.34% versus 1.49 ± 0.73%; Fig 2D and E). Taken together, the above data demonstrated that after sCreER was switched to Cre, it targeted recombination more efficiently than conventional CreER for some genomic DNAs that are known to be less susceptible to recombination. Figure 2. sCreER has significantly higher recombination efficiency than CreER by R26-Confetti reporter A. Schematic diagram showing recombination readouts by crossing of Npr3-CreER or Npr3-sCreER with R26-Confetti reporter. B. Schematic figure showing experimental strategy. 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. 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. Download figure Download PowerPoint To further demonstrate the strength of sCreER in recombination on different reporters, we used two additional reporter alleles for comparison of the recombination efficiency of sCreER and CreER. We first generated a new reporter R26-loxP-ZsGreen-loxP-tdTomato (named as R26-LZLT), in which long DNA sequence containing Stop-ZsGreen-polyA was flanked by two loxP sites (Fig 3A). We crossed Npr3-sCreER or Npr3-CreER with R26-LZLT, induced tamoxifen at E11.5, and collected hearts at E14.5-E15.5 for analysis (Fig 3B). We found that the percentage of endocardial cells expressing tdTomato was significantly higher in Npr3-sCreER;R26-LZLT than Npr3-CreER;R26-LZLT (Fig 3C–E). Additionally, we crossed Npr3-sCreER or Npr3-CreER with R26-GFP reporter (Daigle et al, 2018; Zhao et al, 2018) and found that Npr3-sCreER was far more efficient in recombining R26-GFP than Npr3-CreER (Appendix Fig S6). Taken together, the above data demonstrated that sCreER was more efficient than CreER in recombination of a refractory target locus. Figure 3. Recombination efficiency by sCreER or CreER on R26-LZLT reporter Schematic diagram showing Npr3-sCreER or Npr3-CreER mouse crossed with a newly generated Rosa26 reporter R26-LZLT. Schematic diagram showing experimental strategy. 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. Representative immunostaining images for tdTomato, ESR, and CDH5 on heart sections. 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. Download figure Download PowerPoint sCreER deletes genes more efficiently than CreER Inadequate gene deletion by CreER resulting from inefficient recombination on some inert genomic loci represents a major hurdle for its in vivo use. To test whether sCreER recombines DNA more efficiently for in vivo gene deletion, we crossed Npr3-sCreER with Kdr-flox allele that encodes VEGFR2, and treated the offspring mice with Tam to induce Cre-loxP recombination (Fig 4A). We adopted the same Tam treatment strategy to analyze the deletion efficiency of Kdr gene by immunostaining for the VEGFR2 protein (Fig 4B). Without Tam, VEGFR2 could be robustly detected in endocardial cells of the developing heart (Fig 4C). After Tam, VEGFR2 could still be detected in most endocardial cells of atria and ventricles of Npr3-CreER;Kdrflox/flox mice when compared with that of Npr3-CreER;Kdr+/+ or Npr3-CreER;Kdrflox/+ littermate controls (Fig 4D), indicating that the conventional CreER did not efficiently knockout the Kdr gene. In contrast, VEGFR2 could not be detected in most endocardial cells in atria and ventricles of the Npr3-sCreER;Kdrflox/flox mice (Fig 4E). As internal control, VEGFR2 was found present in the Npr3-sCreER;Kdr+/+ or Npr3-sCreER;Kdrflox/+ littermates (Fig 4E). Figure 4. Increased efficiency of gene deletion by sCreER, compared with CreER A. Schematic diagram showing Kdr gene deletion by sCreER. B. Schematic figure showing experimental design. C. Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam). D, E. Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium. 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. Download figure Download PowerPoint To further validate this finding, we generated R26-tdTomato;Kdr-flox mice to compare the recombination efficiency between different alleles using sCreER or CreER in the same mouse (Fig 4F and G). In these mice, tdTomato expression or the lack of VEGFR2 expression in endocardial cells indicates efficient Cre-loxP recombination in the R26-tdTomato or Kdr-flox allele, respectively. In the mouse hearts, VEGFR2 was found expressed in endocardial cells located in the trabecular myocardium (TM) and also in the coronary endothelial cells located in compact myocardium (CM) region (Zhang et al, 2016c). Npr3-CreER efficiently labeled endocardial cells by tdTomato, but VEGFR2 was not deleted in the majority of tdTomato+CDH5+ endocardial cells of TM of Kdrflox/flox and Kdrflox/+ hearts (Fig 4F). In contrast, Npr3-sCreER not only robustly labeled endocardial cells by tdTomato, but also efficiently ablated Kdr gene in endocardial cells of TM by Kdrflox/flox when compared with Kdrflox/+ (Fig 4G). In the endocardial cells of TM, the percentage of tdTomato+ endocardial cells that expressed VEGFR2 in Npr3-sCreER;Kdrflox/+ and Npr3-sCreER;Kdrflox/flox mice was 91.94 ± 2.30% and 11.18 ± 2.05%, respectively (Fig 4G). Similarly, the percentage of CDH5+ endocardial cells that expressed VEGFR2 in Npr3-sCreER;Kdrflox/+ and Npr3-sCreER;Kdrflox/flox mice was 96.34 ± 1.07% and 15.12 ± 2.20%, respectively (Appendix Fig S7). Notably, VEGFR2 expression was not detected in most endocardial cells of TM in Npr3-sCreER;Kdrflox/flox mice; it could still be observed in the coronary endothelial cells of CM (Fig 4G), indicating that the specificity of sCreER under Npr3 promoter targeted endocardial cells but not coronary endothelial cells. Taken together, these data demonstrated that sCreER could self-cleave and switch to Cre for robust gene deletion. Generation and characterization of Col1a2-sCreER and Col1a2-CreER alleles To independently test the self-cleavage property in different mouse lines, we also generated two additional knock-in alleles on the Col1a2 gene locus: Col1a2-sCreER and Col1a2-CreER by utilizing the same targeting strategies via CRISPR/Cas9 (Fig 5A). We respectively crossed them with the R26-tdTomato reporter line, inducted tamoxifen at E12.5, and collected hearts for analysis at E14.5-E15.5 (Fig 5B). Consistent with previous studies on the Col1a2 gene's specificity (van Amerongen et al, 2008; He et al, 2017), both Cre lines robustly labeled the PDGFRa+ fibroblast cell lineages in the valve mesenchyme and epicardial cells (Fig 5C and D). Immunostaining for tdTomato and ESR on heart sections showed that while ESR was expressed in tdTomato+ cells of the Col1a2-CreER;R26-tdTomato hearts, ESR was hardly detected in most tdTomato+ cells of the Col1a2-sCreER;R26-tdTomato hearts (Fig 5E). Quantification data showed that the labeling efficiency by tdTomato was comparable between Col1a2-sCreER and Col1a2-CreER (Fig 5F). However, the percentag
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