Cre recombinase resources for conditional mouse mutagenesis

Large scale international activities for systematic conditional mouse mutagenesis, exploiting advances in the sophisticated manipulation of the mouse genome, has established the mouse as the premier organism for developing models of human disease and drug action. Conditional mutagenesis is critical for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal. The majority of new mouse mutants are therefore designed as conditional, activated only in a specific tissue (spatial control) and/or life stage (temporal control) through biogenic Cre/loxP technologies. The full power of conditional mutant mice can therefore only be exploited with the availability of well characterized mouse lines expressing Cre-recombinase in tissue, organ and cell type-specific patterns, to allow the creation of somatic mutations in defined genes. This chapter provides an update on the current state of Cre driver mouse lines worldwide, and reviews the available public databases and portals that capture critical details of Cre driver lines such as the efficiency of recombination, cell tissue specificity, or genetic background effects. The continuously changing landscape of these mouse resources reflects the rapid progression of research and development in conditional and inducible mouse mutagenesis.     

Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissues

Here we describe the generation of the Nes-Cre1 transgenic mouse line in which Cre recombinase expression is controlled by the rat nestin promoter and intron 2 enhancer. This line has previously been used for conditional loss-of-function studies of various genes in the central nervous system and first branchial arch ectoderm. Here we report the detailed temporal and spatial recombination pattern of Nes-Cre1 using three different reporters of Cre-mediated recombination, ROSA26R (R26R), Z/AP, and Z/EG. Cre/loxP recombination was detected in embryos as early as the head-fold stage. By embryonic day (E)15.5 recombination occurred in virtually all cells of the nervous system and unexpectedly also in somite-derived tissues and kidneys. Tissues with little or no recombination included heart, liver, thymus, and lung. This study suggests that Nes-Cre1-mediated recombination occurs in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites.     

Use of ERT2-iCre-ERT2 for conditional transgenesis

We examined the use of ERT2-iCre-ERT2 (Cre2ERT2), a tamoxifen-regulated form of Cre that has been described to have a background activity lower than that of other tamoxifen-regulated Cre constructs, for establishing performant conditional deleter mouse lines. Cre2ERT2 was inserted by homologous recombination into the Rosa26 locus. These mice were mated with R26R Cre-reporter mice. No recombination could be observed in the progenies in the absence of tamoxifen treatment. Tamoxifen treatment at E13-14 led to a high level, albeit variable, recombination in most of the tissues examined: liver, heart, kidney, brain, lung etc. Treatment of adult animals also induced recombination in these tissues, although at a lower level. Northern blot and qPCR studies suggested that these differences are not linked to significant variations of the level of expression of Cre2ERT2. Thus, Cre2ERT2 appears to be a good alternative to existing modulatable Cre systems, displaying a lack of background activity and a high-level inducibility in vivo.     

Tamoxifen for induction of Cre-recombination may confound fibrosis studies in female mice

A variety of conditional knock-out mice relying on Tamoxifen-driven ERT2/Cre -mediated recombination are available and have been used to study involvement of specific genes in kidney disease. However, recent data suggest that Tamoxifen itself might attenuate fibrosis when administered during experimental models of kidney disease. It has remained unclear whether this still applies also if kidney damage is initiated after a wash-out period has been implemented. Here we report that the commonly applied regimen of administration of 4 alternate day doses of 1mg Tamoxifen per mouse until 14 days prior to start of the actual experiment, in this case the induction of obstructive nephropathy by Unilateral Ureteral Obstruction (UUO), still attenuated fibrosis in female obstructed mouse kidneys, whereas this effect was not seen in male obstructed kidneys. Attenuation of fibrosis was accompanied by a reduction in nuclear ERα positivity despite absence of detectable levels of the active tamoxifen metabolite endoxifen throughout the UUO experiment. In conclusion, these results indicate that the Tamoxifen dosing regimen commonly applied in conditional gene targeting experiments might have prolonged confounding effects in female mice through attenuation of renal fibrosis independent of modulation of the expression of the targeted gene(s).     

Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.     

A nested deletion approach to generate Cre deleter mice with progressive Hox profiles

In mice, the loxP/Cre recombinase-dependent system of recombination offers powerful possibilities for engineering genetic configurations of interest. This system can also be advantageously used for conditional mutagenesis in vivo, whenever such an approach is required due to deleterious effects of either one mutation, or a combination thereof. Here, we report on the production of an allelic series of insertions of a Hoxd11/Cre fusion transgene at different positions within the HoxD complex, in order to produce the CRE recombinase with a 'Hox profile' progressively more extended. We used the R26R (R26R) reporter mouse line to functionally assess the distribution and efficiency of the CRE enzyme and discuss the usefulness of these various lines as deleter strains.     

Temporally and spatially regulated somatic mutagenesis in mice.

In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the earliest phenotype of the introduced mutation can eclipse later effects. We show in mice that site-specific genomic recombination can be induced in a selected cell type, B lymphocytes, at a chosen time. This precision of somatic mutagenesis was accomplished by limiting expression of a Cre recombinase-estrogen receptor fusion protein to B lymphocytes by use of tissue-specific elements in the promoter of the transgene employed. The expressed fusion protein remained inactive until derepressed by systemic administration of an exogenous ligand for the estrogen receptor, 4-OH-tamoxifen. Upon derepression the Cre recombinase enzyme deleted specific DNA segments, flanked by loxP sites, in B lymphocytes only. The efficiency of recombination in cells expressing the fusion protein could be varied from low levels to >80%, depending on the dose of ligand administered. Our work presents a paradigm applicable to other uses of site-specific recombination in somatic mutagenesis where both temporal and spatial regulation are desired.     

Site- and time-specific gene targeting in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.     

VavCre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages

Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific somatic tissues. We have generated vavCre transgenic mice, which can be used to inactivate genes specifically in adult hematopoietic and endothelial cells. In these animals, a Cre transgene is expressed under control of murine vav gene regulatory elements. To assess their usefulness, vavCre transgenic mice were bred with R26R mice, which express a lacZ reporter gene only in cells where Cre-mediated recombination has occurred. VavCre/R26R double-heterozygous offspring were analyzed by beta-galactosidase histochemistry and flow cytometry. VavCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs, including the hematopoietic progenitor-rich bone marrow. Recombination also occurred in most endothelial and germ cells, but only rarely in other cell types. The recombination in both hematopoietic and endothelial lineages may partly reflect their putative shared ontogeny and provides a unique tool for simultaneous pan-hematopoietic and endothelial mutagenesis.     

Conditional transgenesis using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCrexR26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.     

Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.     

Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements

Summary: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions. In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells. The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ER(T2) and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks. High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line. Critically, no EGFP expression was observed in any uninduced mice. This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner. genesis 33:191-197, 2002.     

Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.     

Targeted insertion of an IRES Cre into the Hnf4alpha locus: Cre-mediated recombination in the liver, kidney, and gut epithelium

The Hnf4alpha gene belongs to a family of trancriptional regulators required for liver development and function. Hnf4alpha is also expressed in other tissues, including the newly formed visceral endoderm of the early postimplantation embryo, and later in embryogenesis in the gut epithelium and the kidney. The regulatory sequences involved in controlling expression of Hnf4alpha at these diverse sites are not clearly understood. Here we used homologous recombination to introduce Cre recombinase coding sequences into the endogenous Hnf4alpha locus. Crossing Hnf4alpha(Creex2/+) mice with R26R partners allowed us to follow the pattern of Cre-mediated recombination. Our results show that recombination of the reporter allele closely follows endogenous Hnf4alpha expression, but with a slight temporal delay. Thus, the Hnf4alpha(Creex2) strain should prove useful for conditionally deleting gene activity in the liver, gut epithelium, or kidney.     

Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice

Conditional mutagenesis permits the cell type-specific analysis of gene functions in vivo. Here, we describe a mouse line that expresses Cre recombinase under control of regulatory sequences of NEX, a gene that encodes a neuronal basic helix-loop-helix (bHLH) protein. To mimic endogenous NEX expression in the dorsal telencephalon, the Cre recombinase gene was targeted into the NEX locus by homologous recombination in ES cells. The Cre expression pattern was analyzed following breeding into different lines of lacZ-indicator mice. Most prominent Cre activity was observed in neocortex and hippocampus, starting from around embryonic day 11.5. Within the dorsal telencephalon, Cre-mediated recombination marked pyramidal neurons and dentate gyrus mossy and granule cells, but was absent from proliferating neural precursors of the ventricular zone, interneurons, oligodendrocytes, and astrocytes. Additionally, we identified formerly unknown domains of NEX promoter activity in mid- and hindbrain. The NEX-Cre mouse will be a valuable tool for behavioral research and the conditional inactivation of target genes in pyramidal neurons of the dorsal telencephalon.     

Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells

Continuous expression of Cre recombinase has the potential to yield toxic side effects in various cell types, thereby limiting applications of the Cre/loxP system for conditional mutagenesis. In this study, we investigate the potential of Cre protein transduction to overcome this limitation. COS-7, CV1-5B, and mouse embryonic stem (ES) cells treated with cell-permeant Cre (HTNCre) maintain a normal growth behavior employing Cre concentrations sufficient to induce recombination in more than 90% of the cells, whereas continuous application of high doses resulted in markedly reduced proliferation. HTNCre-treated ES cells maintain a normal karyotype and are still able to contribute to the germline. Moreover, we present an enhanced HTNCre purification protocol that allows the preparation of a concentrated glycerol stock solution, thereby enabling a considerable simplification of the Cre protein transduction procedure. The protocol described here allows rapid and highly efficient conditional mutagenesis of cultured cells.