The Cre/lox system: Cre-ating unintended damage
2019; American Physical Society; Volume: 316; Issue: 5 Linguagem: Inglês
10.1152/ajprenal.00428.2018
ISSN1931-857X
Autores Tópico(s)Photosynthetic Processes and Mechanisms
ResumoEditorial FocusThe Cre/lox system: Cre-ating unintended damageLeslie S. GewinLeslie S. GewinDivision of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Medicine, Veterans Affairs Hospital, Tennessee Valley Healthcare System, Nashville, Tennessee; and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TennesseePublished Online:29 Apr 2019https://doi.org/10.1152/ajprenal.00428.2018This is the final version - click for previous versionMoreSectionsPDF (37 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat INTRODUCTIONOne of the pivotal advances in molecular biology has been the ability to target mammalian gene expression in a site-specific manner. This is achieved using the Cre/lox technique in which the enzyme cyclization recombinase (Cre) from the bacteriophage P1 recognizes certain 34-bp loxP sites and removes intervening DNA through recombination (8, 9). Thus, a murine gene (or key exon) of interest can be flanked by loxP sites and removed by Cre recombinase (2). Spacial specificity is obtained by putting the expression of Cre under the control of specific promoters that are only active in the cell type of interest, and inducible promoters allow for temporal control of Cre-mediated recombination. This Cre/lox system has been important for defining the function of proteins in different mammalian tissues (7) and for lineage tracing studies when used with a reporter mouse containing a stop codon flanked by loxP sites.EXPRESSION OF CRE AND GLOMERULAR PATHOLOGYThe Cre enzyme itself has long been assumed to be inactive in the absence of a gene flanked by loxP sites (i.e., floxed), and most investigators will used the floxed littermates without Cre as controls. In an article published in a recent issue of the American Journal of Physiology-Renal Physiology, Balkawade et al. (1) showed that uninjured mice with podocyte-specific expression of Cre (NPHS2-Cre) have subtle but definite glomerular pathology. There were no gross functional differences between NPHS2-Cre mice or wild-type mice as measured by proteinuria and creatinine, consistent with the lack of pathology previously reported (5). More thorough investigation by the authors revealed that the number of cells staining for Wilms tumor 1 (WT1), a marker of podocytes, was decreased in NPHS2-Cre+/+ mice at 24 wk of age. Additionally, statistically significant differences in glomerular basement membrane (GBM) thickness, as measured by electron microscopy, were noted between wild-type (186.8 ± 3.6 nm) versus NPHS2-Cre+/− (236.9 ± 6.5 nm) or NPHS2-Cre+/+ (256.8 ± 10 nm) mice (1).To investigate the mechanism whereby NPHS2-Cre increased GBM thickness, the authors examined protein and transcriptional regulation of the individual laminin chains that comprise mature laminin-α5β2γ1 (LAM-521). Both α5- and β2-chains were suppressed, whereas γ1-chains were increased, in NPHS2-Cre+/+ compared with wild-type mice. This would be consistent with a switch to immature laminin-α1β1γ1 (LAM-111) but does not prove this as α1- and β1-chain levels were not assessed. Also, it is curious that NPHS2-Cre+/− mice actually had less γ1 protein expression than wild-type mice despite increased GBM thickness, making this explanation less likely. To further probe these NPHS2-Cre-dependent changes in podocytes and GBM thickness, RNA sequencing was performed on isolated glomeruli of NPHS2-Cre+/+ and wild-type mice. There were 230 genes with statistically significant alterations in expression between the 2 groups, including 15 matrix-associated genes and 17 apoptosis/cell death-related genes. Some of the genes upregulated in NPHS2-Cre mice have antifibrotic roles (e.g., cytoglobin), indicating that many of these transcripts are altered in response to injury rather than as the cause. Although the number of mice used for RNA sequencing was low (2 mice/genotype), this brief report carefully documents structural changes in the glomeruli of NPHS2-Cre mice and associated alterations in genes involved in matrix production and cell survival (1).It is reasonable to question the significance of these changes in GBM thickness and WT1+ cells given that no major differences in proteinuria or renal function were detected. Aside from the issue that creatinine is notoriously insensitive as a measure of renal function, it is important to recognize that mice generated by the Cre/lox technique are often used to assess the cell-specific role of a protein in renal injury. Relatively subtle pathology in uninjured mice can predispose to greater susceptibility to injury, and an observed phenotype may be caused by Cre-dependent effects and not solely by conditional deletion of the targeted gene. This is the first report to document Cre-dependent damage in the kidney, but injury in other organs has been previously published. Using the α-myosin heavy chain (α-MyHC) promoter to drive Cre expression in the heart, investigators have reported a reduction in cardiac function at 6 mo with fibrosis, inflammation, and DNA damage (6). Similarly, targeting lung epithelia using Cre under control of the human surfactant protein C promoter caused cystic lungs with excessive apoptosis (3). The phenotypes of extrarenal Cre activity were more pronounced than that of NPHS2-Cre, but DNA damage and epithelial apoptosis appear to be common Cre-mediated pathologies. Although NPHS2-Cre-mediated apoptosis was not shown directly, the decrease in WT1+ cells in Cre-containing mice and the increase in transcripts of DNA damage/apoptosis genes by RNA sequencing is suggestive (1).The large difference in phenotype severity between NPHS2-Cre and that published in the heart and lungs raises the questions of why does this variability occur? The amount and duration of Cre expression, which varies based on the promoter, may account for these differences. Consistent with this idea, much of the cardiac toxicity induced by α-MyHC-Cre was averted by transient induction of Cre using a tamoxifen-inducible α-MyHC promoter (4). Another question raised in Balkawade et al.’s work is how does Cre cause this unintended injury? Although Cre preferentially mediates recombination between pairs of loxP sites, it can induce recombination in endogenous sites that have up to 10 mismatches to the canonical 34-bp loxP (10). These cryptic or degenerate loxP sites were identified in transcriptionally active cardiac genes, and changes in transcription and protein expression of these genes were noted when α-MyHC-Cre was present (6). Another possible explanation is that transgene insertion site or copy number could mediate both the phenotype variability and Cre-mediated injury. Although not investigated with NPHS2-Cre, this possibility was considered less likely with the Cre-mediated cardiotoxicity based on insertion site mapping (6).IMPLICATIONSSo, what are the implications of this study for the field of renal injury and the Cre/lox system? The obvious answer is that Cre-containing mice without the floxed alleles should also be used as controls. This approach requires increasing the mouse colony costs as additional breeders would be necessary. It is important to maintain equivalent background strains between Cre-containing control and Cre plus floxed allele mice as there is variability in Cre-dependent effects based on genetic background (4). Adding additional controls to costly mouse experiments, in addition to the National Institutes of Health mandate to study both sexes, can be a hard pill to swallow, particularly when the budgets for studies are becoming more restrictive. Certainly, there is a case to be made for increasing the budgets for models of injury using genetically altered mice. However, ignoring the direct effect of Cre itself has even more costly consequences as the Cre/lox system is used to generate important preclinical data for potential therapeutics.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author(s).AUTHOR CONTRIBUTIONSL.G. drafted manuscript; L.G. edited and revised manuscript; L.G. approved final version of manuscript.REFERENCES1. Balkawade RS, Chen C, Crowley MR, Crossman DK, Clapp WL, Verlander JW, Marshall CB. Podocyte-specific expression of Cre recombinase promotes glomerular basement membrane thickening. Am J Physiol Renal Physiol. doi:10.1152/ajprenal.00359.2018. Link | ISI | Google Scholar2. Gu H, Zou YR, Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73: 1155–1164, 1993. doi:10.1016/0092-8674(93)90644-6. Crossref | PubMed | ISI | Google Scholar3. Jeannotte L, Aubin J, Bourque S, Lemieux M, Montaron S, Provencher St-Pierre A. Unsuspected effects of a lung-specific Cre deleter mouse line. Genesis 49: 152–159, 2011. doi:10.1002/dvg.20720. Crossref | PubMed | ISI | Google Scholar4. Lexow J, Poggioli T, Sarathchandra P, Santini MP, Rosenthal N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver. Dis Model Mech 6: 1470–1476, 2013. doi:10.1242/dmm.010470. Crossref | PubMed | ISI | Google Scholar5. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB. Podocyte-specific expression of cre recombinase in transgenic mice. Genesis 35: 39–42, 2003. doi:10.1002/gene.10164. Crossref | PubMed | ISI | Google Scholar6. Pugach EK, Richmond PA, Azofeifa JG, Dowell RD, Leinwand LA. Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic. J Mol Cell Cardiol 86: 54–61, 2015. doi:10.1016/j.yjmcc.2015.06.019. Crossref | PubMed | ISI | Google Scholar7. Rossant J, Nagy A. Genome engineering: the new mouse genetics. Nat Med 1: 592–594, 1995. doi:10.1038/nm0695-592. Crossref | PubMed | ISI | Google Scholar8. Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 85: 5166–5170, 1988. doi:10.1073/pnas.85.14.5166. Crossref | PubMed | ISI | Google Scholar9. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150: 467–486, 1981. doi:10.1016/0022-2836(81)90375-2. Crossref | PubMed | ISI | Google Scholar10. Thyagarajan B, Guimarães MJ, Groth AC, Calos MP. Mammalian genomes contain active recombinase recognition sites. Gene 244: 47–54, 2000. doi:10.1016/S0378-1119(00)00008-1. Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: L. S. Gewin, Vanderbilt University Medical Center, Rm. S3304 MCN, 1161 21st Ave. S, Nashville, TN 37232 (e-mail: l.[email protected]org). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Related ArticlesPodocyte-specific expression of Cre recombinase promotes glomerular basement membrane thickening 08 May 2019American Journal of Physiology-Renal PhysiologyCited ByEffect of Thymoquinone on Renal Damage Induced by Hyperlipidemia in LDL Receptor-Deficient (LDL-R-/-) MiceBioMed Research International, Vol. 2022Glutamate homeostasis and dopamine signaling: Implications for psychostimulant addiction behaviorNeurochemistry International, Vol. 144Cre recombinase toxicity in podocytes: a novel genetic model for FSGS in adolescent miceMadeleine Frahsek,* Kevin Schulte,* Arnaldo Chia-Gil, Sonja Djudjaj, Herdit Schueler, Katja Leuchtle, Bart Smeets, Henry Dijkman, Jürgen Floege, and Marcus J. Moeller7 November 2019 | American Journal of Physiology-Renal Physiology, Vol. 317, No. 5 More from this issue > Volume 316Issue 5May 2019Pages F873-F874 https://doi.org/10.1152/ajprenal.00428.2018PubMed30943071History Received 1 March 2019 Accepted 21 March 2019 Published online 29 April 2019 Published in print 1 May 2019 Metrics
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