Cre‐<i>lox</i>P‐Mediated Recombination: General Principles and Experimental Considerations

McLellan, Micheal A.; Rosenthal, Nadia; Pinto, Alexander R.

doi:10.1002/cpmo.22

Cited by 137 publications

(91 citation statements)

References 78 publications

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“…Arguably one of the most important technological advancements in the study of gene function has been the development of gene "knock‐outs" and "knock‐ins." Furthermore, the conditional targeting by cell or tissue specific promoters using the Cre/loxP system has been instrumental in elucidating gene function in specific cell populations in vivo (reviewed in [1]). Further sophistication to this system was added by allowing temporal control by fusing the Cre recombinase to a mutated ligand binding domain of the estrogen receptor.…”

Section: Figurementioning

confidence: 99%

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Stifter

Greter

2020

Eur J Immunol

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Cre and CreER mouse strains are powerful tools that have proven invaluable for investigating the function of genes and for the fate‐mapping of cell populations. The fidelity of these systems however are becoming more and more contested. In this issue of the European Journal of Immunology, Van Hove et al. and Chappell‐Maor et al. carefully dissect the cellular specificities of two commonly used CreER mouse strains for the study of CNS macrophages; Cx3cr1CreER and Sall1CreER. Both studies elegantly highlight that CreER strains, as well as the "floxed" allele to be targeted, need to be carefully selected and properly characterized in order to ensure reproducible and robust data and interpretations. These studies are a cautionary tale for this technology, but also highlight that we must continuously question and improve our experimental approaches.

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Section: Figurementioning

confidence: 99%

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Stifter

Greter

2020

Eur J Immunol

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“…While the allele is capable of recombination, we observed variable efficiency that appeared to be dependent on the cell type. While this could simply reflect variable expression or activity of individual Cre transgenes or recombinase, respectively (Bao, Ma, Schuster, Lin, & Yan, ; McLellan, Rosenthal, & Pinto, ), we hypothesize that a cis effect at the Irf6 locus contributes to the variable efficiency. This hypothesis is based on the following: (1) In this study, we observed no or incomplete recombination with the EIIa‐Cre “deleter strain,” and with two other tissue specific Cre transgenes, including K14‐Cre‐ER (Vasioukhin, Degenstein, Wise, & Fuchs, ) and Tgfb3‐Cre (Yang, Li, & Kaartinen, ; data not shown).…”

Section: Resultsmentioning

confidence: 80%

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Smith

Kousa

Kinoshita

et al. 2017

Genesis

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Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used.

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“…The CreERT2 system, in combination with floxed genes allows visualization of cells, overexpression or suppression of their genes, cell ablation, inhibition, and excitation [36,37]. By combining mural cell promoter lines with fluorescent reporter lines, we enabled the visualization of individual mural cells for detailed morphological characterization.…”

Section: Mural Cell-specific Creert2 Lines For Basic Researchmentioning

confidence: 99%

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow

Ivanova

Corona

Eleftheriou

et al. 2020

Preprint

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Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. Across three lines, under either the PDGFRb or NG2 promoter, the specificity and pattern of Cre activation varied significantly. In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes.Furthermore, in one PDGFRb line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficit, a common pathology in numerous diseases. *

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Cre‐loxP‐Mediated Recombination: General Principles and Experimental Considerations

Cited by 137 publications

References 78 publications

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow

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