CreER^T2 expression from within the c‐Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells

Abstract

Mast cells are abundantly situated at contact sites between the body and its environment, such as the skin and, especially during certain immune responses, at mucosal surfaces. They mediate allergic reactions and degrade toxins as well as venoms. However, their roles during innate and adaptive immune responses remain controversial and it is likely that major functions remain to be discovered. Recent developments in mast cellspecific conditional gene targeting in the mouse promise to enhance our understanding of these fascinating cells. To complete the genetic toolbox to study mast cell development, homeostasis and function, it is imperative to inducibly manipulate their gene expression. Here, we report the generation of a novel knock-in mouse line expressing a tamoxifeninducible version of the Cre recombinase from within the endogenous c-Kit locus. We demonstrate highly efficient and specific inducible expression of a fluorescent reporter protein in mast cells both in vivo and in vitro. Furthermore, induction of diphtheria toxin A expression allowed selective and efficient ablation of mast cells at various anatomical locations, while other hematopoietic cells remain unaffected. This novel mouse strain will hence be very valuable to study mast cell homeostasis and how specific genes influence their functions in physiology and pathology. Keywords: c-Kit locus r Inducible conditional gene targeting r Mast cellsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMast cells belong to the innate arm of the immune system and mediate immunoglobulin E (IgE)-associated reactions. CrossCorrespondence: Dr. Marc Schmidt-Supprian e-mail: supprian@biochem.mpg.de linking of their high affinity IgE receptor (FcεRI) leads to secretion of preformed granule associated mediates such as vasoactive amines and proteases, and to de novo synthesis of proinflammatory lipid mediators and cytokines [1]. In addition, several reports * These authors contributed equally to this work.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 296-306 New technology 297 attest mast cells numerous IgE-independent effector and regulatory functions in innate and adaptive immune responses against bacteria and pathogens [2-4], autoimmunity [5][6][7], tolerance induction [8][9][10], obesity and diabetes [11], contact hypersensitivity [12], angiogenesis, and malignant diseases [13]. Most in vivo evidence for these functions relies on the use of mice lacking mast cells mainly due to various spontaneous loss-of-function mutations in the white-spotting (W) locus that encodes for the receptor tyrosine kinase c-Kit [14][15][16][17]. However, recent experiments using c-Kit-independent mast cell-deficient mice have failed to corroborate some of these initial findings [18][19][20]. While the exact reasons for these discrepant results remain unclear, it appears obvious that the further elucidation of in vivo mast cell functions will benefit from novel...

Absence of Evidence Is Not Evidence of Absence

“…13,14 Remarkably, using similar strategies as van Berlo et al 1 for Cre-dependent recombination by tamoxifen, this mouse shows that this protocol recombines <3% of the Sca-1+/c-kit+ HSCs. Even the efficiency of recombination in mast cells, despite expressing c-kit (and Cre) at many fold higher level than the HSCs, 14 shows heterogeneous rates of recombination from tissue to tissue.…”

“…13,14 Remarkably, using similar strategies as van Berlo et al 1 for Cre-dependent recombination by tamoxifen, this mouse shows that this protocol recombines <3% of the Sca-1+/c-kit+ HSCs. Even the efficiency of recombination in mast cells, despite expressing c-kit (and Cre) at many fold higher level than the HSCs, 14 shows heterogeneous rates of recombination from tissue to tissue. Thus, this mouse model, and by extension all those that depend on the strength of the c-kit promoter to drive Cre, is not adequate for tracking the cell fates of adult multipotent stem and progenitors cells expressing intermediate or low levels of c-kit and where the indispensable function of the receptor allows only hemizygosis of the knocked in Cre allele.…”

“…Thus, this mouse model, and by extension all those that depend on the strength of the c-kit promoter to drive Cre, is not adequate for tracking the cell fates of adult multipotent stem and progenitors cells expressing intermediate or low levels of c-kit and where the indispensable function of the receptor allows only hemizygosis of the knocked in Cre allele. Furthermore, in the articles by Klein et al 13 and Heger et al, 14 the undisputable dependence of recombination rate on the level of Cre expression was used to ablate the mast cells and their precursors selectively (or intestinal Cajal cells) without affecting other c-kit-expressing cells (low/intermediate), including the HSCs. These results further emphasize the need to demonstrate a substantial and reproducible level of recombination in the target cells (in this case the eCSCs) in response to the activating protocol used.…”

“…However, the level of c-kit expression by the c-kit+ eCSCs in several strains of mice tested is significantly lower than in the HSCs (Torella et al, unpublished data, 2014). As the c-kit Cre T2 knockin (with its higher affinity for tamoxifen and higher recombination efficiency) recombines <10% of the HSCs (even after a longer pulse with tamoxifen-added chow), 14 it is likely that an even lower fraction, if any, of c-kit+ eCSCs are recombined in the Cre/lox strategy of Molkentin's group. Independent of the correctness of the above conclusion, it is indisputable that the efficiency of recombination in the ckit+ eCSCs, which is the main subject of the article, should have been determined before attempting to evaluate their contribution to the myocardium using a model that is completely dependent on the efficiency of recombination to turn on the expression of the reporter gene.…”

“…Clearly, these caveats should have been experimentally addressed particularly given the significance of the data discussed above and the fact that many bona fide c-kit+ cells do not recombine. 13,14 There are 2 additional issues that might also affect the interpretation of the results of van Berlo et al 1 First, the inner cell mass cells of the blastocyst (ie, the embryonic stem cells) express intermediate but significant levels of c-kit. 7,15,16 The level of c-kit expression is even higher in the primordial germ cells.…”