A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse

Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted. Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced, be affected by position-effect variegation, or reside in a chromatin configuration inaccessible for recombination. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites, and the availability of both wild-type (WT) and mutant loxP sites, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.     

Conditional cell ablation via diphtheria toxin reveals distinct requirements for the basal plate in the regional identity of diencephalic subpopulations

The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1–p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen‐ induced, Cre‐mediated recombination of the ROSA^DTA allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate‐deficient embryos, the p1 alar plate exhibited reduced expression of subtype‐specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments. genesis 53:356–365, 2015. © 2015 Wiley Periodicals, Inc.     

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh‐Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α‐smooth muscle actin‐labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β‐HSD double‐positive fetal LCs could be observed in Amh‐Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β‐HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs’ central role in fetal testis development.     

Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~ E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.     

Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice

Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient–rather than permanent–inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreER^T2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12–30 days post-TAM displayed indices of a stress-induced anxiety phenotype–longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype–longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a depressive phenotype, and recovery of DCX+ cell number corresponds to normalization of these behaviors.     

A novel reporter rat strain that expresses LacZ upon Cre‐mediated recombination

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP‐dependent oncogenic H‐ or K‐ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre‐expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.     

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts

Background information. N‐cadherin, a member of the Ca²⁺‐dependent cell—cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N‐cadherin‐dependent cell—cell contacts in myoblasts remain unexplored. Results. In the present study, we show that N‐cadherin‐dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N‐cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N‐cadherin‐dependent cell contacts, characterized by the immobilization of a pool of N‐cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F‐actin filaments. We also demonstrated that the formation of N‐cadherin‐dependent cell—cell contacts requires a co‐ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N‐cadherin‐dependent cell—cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N‐cadherin‐dependent cell contact formation, since, via ROCK (Rho‐associated kinase) signalling and myosin 2 activation, it allows the stabilization of N‐cadherin at the cell—cell contact sites. Conclusions. We have shown that Rac1 and RhoA have opposite effects on N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.     

Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1lox/lox and Stra8‐iCre; Mov10l1lox/Δ mice

In the Cre–loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre‐mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8‐codon‐improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell‐specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre‐mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre–loxp system is used for deleting genes in a specific cell lineage and the Cre; gene^lox/Δ genotype should be used to evaluate phenotypes instead of Cre; gene^lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 51:481–490. © 2013 Wiley Periodicals, Inc.     

Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement

Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.     

A multifunctional reporter mouse line for Cre‐ and FLP‐dependent lineage analysis

The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26^NZG) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site‐of‐integration effects often observed with transgenic reporters. R26^NZG directs Cre‐dependent nuclear‐localized β‐galactosidase (β‐gal) expression, and can be converted into a Cre‐dependent EGFP reporter (R26^NG) by germline excision of the FRT‐flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26^NZG generates an FLP‐dependent EGFP reporter (R26^ZG) that expresses β‐gal in FLP‐nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26^NZG allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems. genesis 47:107–114, 2009. © 2009 Wiley‐Liss, Inc.     

Rapamycin treatment dose‐dependently improves the cystic kidney in a new ADPKD mouse model via the mTORC1 and cell‐cycle‐associated CDK1/cyclin axis

Although translational research into autosomal dominant polycystic kidney disease (ADPKD) and its pathogenesis has made considerable progress, there is presently lack of standardized animal model for preclinical trials. In this study, we developed an orthologous mouse model of human ADPKD by cross‐mating Pkd2 conditional‐knockout mice (Pkd2^f3) to Cre transgenic mice in which Cre is driven by a spectrum of kidney‐related promoters. By systematically characterizing the mouse model, we found that Pkd2^f3/f3 mice with a Cre transgene driven by the mouse villin‐1 promoter (Vil‐Cre;Pkd2^f3/f3) develop overt cysts in the kidney, liver and pancreas and die of end‐stage renal disease (ESRD) at 4–6 months of age. To determine whether these Vil‐Cre;Pkd2^f3/f3 mice were suitable for preclinical trials, we treated the mice with the high‐dose mammalian target of rapamycin (mTOR) inhibitor rapamycin. High‐dose rapamycin significantly increased the lifespan, lowered the cystic index and kidney/body weight ratio and improved renal function in Vil‐Cre;Pkd2^f3/f3 mice in a time‐ and dose‐dependent manner. In addition, we further found that rapamycin arrested aberrant epithelial‐cell proliferation in the ADPKD kidney by down‐regulating the cell‐cycle‐associated cyclin‐dependent kinase 1 (CDK1) and cyclins, namely cyclin A, cyclin B, cyclin D1 and cyclin E, demonstrating a direct link between mTOR signalling changes and the polycystin‐2 dysfunction in cystogenesis. Our newly developed ADPKD model provides a practical platform for translating in vivo preclinical results into ADPKD therapies. The newly defined molecular mechanism by which rapamycin suppresses proliferation via inhibiting abnormally elevated CDK1 and cyclins offers clues to new molecular targets for ADPKD treatment.     

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4‐T‐cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4‐T‐cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T‐cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4‐T‐cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T‐cells involve infection of in vitro activated cells with Cre expressing lenti‐, retro‐, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4‐T‐cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER^T2) under the control of the CD4 gene promoter. We show here that in CD4CreER^T2 mice Cre is inducibly and selectively activated in CD4‐T‐cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4‐T‐cells into effector or suppressor sub‐lineages. genesis 50:908–913, 2012. © 2012 Wiley Periodicals, Inc.     

Melanocytes in conditional Rb–/– mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro

The function of the retinoblastoma tumour suppressor (Rb1), and the pocket protein family in general, has been implicated as an important focal point for deregulation in many of the molecular pathways mutated in melanoma. We have focused on the role of Rb1 in mouse melanocyte homeostasis using gene targeting and Cre/loxP mediated tissue‐specific deletion. We show that constitutive Cre‐mediated ablation of Rb1 exon 2 prevents the production of Rb1 and recapitulates the phenotype encountered in other Rb1 knockout mouse models. Mice with conditional melanocyte‐specific ablation of Rb1 manifest overtly normal pigmentation and are bereft of melanocytic hyperproliferative defects or apoptosis‐induced depigmentation. Histologically, these mice have melanocyte morphology and distribution comparable with control littermates. In contrast, Rb1‐null melanocytes removed from their in vivo micro‐environment and cultured in vitro display some of the characteristics associated with a transformed phenotype. They proliferate at a heightened rate when compared with control melanocytes and have a decreased requirement for mitogens. With progressive culture the cells depigment at relatively early passage and display a gross morphology which, whilst reminiscent of early passage melanocytes, is generally different to equivalent passage control cells. These results indicate that Rb1 is dispensable for in vivo melanocyte homeostasis when its ablation is targeted from the melanoblast stage onwards, however, when cultured in vitro, Rb1 loss increases melanocyte growth but the cells are not fully transformed.     

Non‐parallel recombination limits cre‐loxP‐based reporters as precise indicators of conditional genetic manipulation

Cre/LoxP‐mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage‐tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre‐expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre‐mediated recombination, which raises concerns regarding utilization of Cre‐reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre‐expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non‐parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent‐target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. genesis 51:436–442. © 2013 Wiley Periodicals, Inc.     

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.     

Dissection of gene function at clonal level using mosaic analysis with double markers

MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes--green GFP and red tdTomato -- and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.     

A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.