Cre-mediated recombination in the skin melanocyte lineage

Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.     

Expression of Cre recombinase in pigment cells

Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination.     

Mast cell-specific Cre/loxP-mediated recombination in vivo

Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology. Julia Scholten and Karin Hartmann contributed equally to this work. Supported by grants from the German Research Counsil (Deutsche Forschungsgemeinschaft, RO 2133/2-2) to A.R. and K.H. and the Koeln Fortune Program/Faculty of Medicine, University of Cologne, to A.R. and K.H.. The authors have no conflict of interest     

A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination

The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre-mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.     

Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice

We have established transgenic mice expressing the Cre recombinase under the control of the anti-Müllerian hormone (AMH) gene promoter. Cre activity and specificity were evaluated by different means. In AMH-Cre mice, expression of the Cre recombinase mRNA was confined to the testis and ovary. AMH-Cre mice were crossed with reporter transgenic lines and the offspring exhibited Cre-mediated recombination only in the testis and the ovary. In male, histochemical analysis indicated that recombination occurred in every Sertoli cells. In female, Cre-mediated recombination was restricted to granulosa cells, but the protein was not evenly active in every cells. From these results, we conclude that potentially, this transgenic line possessing AMH promoter-driven expression of the Cre recombinase is a powerful tool to delete genes in Sertoli cells only, in order to study Sertoli cell gene function during mammalian spermatogenesis.     

Thyrocyte-specific expression of Cre recombinase in transgenic mice

A transgenic mouse line that expresses Cre recombinase under control of the human thyroid peroxidase (TPO) gene promoter was established. The activity and specificity of the TPO-driven Cre recombinase were examined by using Northern blotting and by crossing with the ROSA26 reporter transgenic mouse line. In the latter mice, Cre-mediated recombination occurred only in the thyrocytes, and recombination commenced around embryonic day 14.5, at the time during thyroid organogenesis when TPO expression begins. This study demonstrates that the TPO-Cre transgenic mouse is a powerful tool to specifically delete loxP-inserted (floxed) genes in thyrocytes and will be of great value in the study of thyrocyte-specific genes during development and/or in adult thyroids.     

Primary Spermatocyte-Specific Cre Recombinase Activity in Transgenic Mice

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.     

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Tissue-specific and inducible Cre-mediated recombination in the gut epithelium

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine.     

Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination

Conditional alleles containing LoxP recombination sites, in conjunction with Cre recombinase delivered by a variety of means, allows for spatial and temporal control of gene expression in mouse models. Here we describe a mouse strain in which a luciferase (Luc) cDNA, preceded by a LoxP-stop-LoxP (L-S-L) cassette, was introduced into the ubiquitously expressed ROSA26 locus. Mouse embryo fibroblasts derived from this strain expressed luciferase after Cre-mediated recombination in vitro. ROSA26 L-S-L-Luc/+ mice expressed luciferase in a diffuse or liver-restricted pattern, as determined by noninvasive, bioluminescent imaging, when crossed to transgenic mice in which Cre was under the control of a zygotically expressed (EIIA-Cre), or a liver-restricted (albumin-Cre), promoter, respectively. Organ-specific luciferase expression was also seen after intraparenchymal administration of an adenovirus encoding Cre. The ROSA26 L-S-L-Luc/+ strain should be useful for characterizing Cre mouse strains and for following the fate of cells that have undergone Cre-mediated recombination in vivo.     

Direct removal in the mouse of a floxed neo gene from a three-loxP conditional knockout allele by two novel approaches

The presence in an intron of the ploxP-neo-loxP cassette often results in severe interference with gene expression. Consequently, many investigators selectively remove the ploxP-neo-loxP cassette by transient expression of Cre in ES cells. Although effective, the added manipulation of the ES cells may reduce the likelihood that a clone will be able to transmit via the germline. Therefore, we developed two novel approaches that remove the ploxP-neo-loxP by Cre-mediated recombination in mouse. First, the ploxP-neo-loxP-containing mice were crossed with EIIa-Cre transgenic mice. Second, a Cre-expression plasmid was injected into pronuclei of fertilized eggs bearing the ploxP-neo-loxP allele. Both approaches produced mosaic mice with partial and complete excision. These mosaic mice were then mated, and the neo-less conditional knockout allele was found in the offspring after screening only a few litters. These procedures provide options for removing neo directly in the mouse in addition to the commonly used approach that deletes neo in ES cells.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

消化道细胞表达Cre重组酶转基因小鼠的功能鉴定

目的:检测白蛋白启动子介导的Cre重组酶转基因小鼠Alb-Cre-2中Cre重组酶的组织分布及其在体内介导基因重组的作用。方法:将Alb-Cre小鼠与Smad4条件基因打靶小鼠交配,利用PCR对Cre重组酶介导重组的组织特异性进行检测;然后,将Alb-Cre-2转基因小鼠与ROSA26报告小鼠交配,利用LacZ染色对双转基因阳性子代小鼠进行检测。结果:PCR结果显示心、肺、胰、脑及消化道中Cre重组酶介导的Smad4基因发生重组;LacZ染色进一步表明Cre重组酶在肝细胞、胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞、大肠柱状细胞及空泡细胞中特异性表达,并介导ROSA位点LoxP序列间的重组。结论:Alb-Cre-2转基因小鼠在消化道中具有一定的组织特异性,只在胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞,大肠柱状细胞及空泡细胞等细胞类型中特异性表达,并能在体内成功地介导这些消化道上皮细胞基因组上LoxP位点间的重组,是一种研制在消化道特定细胞中特异性基因剔除小鼠的良好工具小鼠。     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

The IRG mouse: a two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ

The Cre-loxP system is widely used for making conditional alterations to the mouse genome. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Given the distinct advantages of fluorescent reporters, we developed a transgenic reporter line, termed IRG, in which DsRed-Express, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and an enhanced green fluorescent protein (EGFP) following recombination. Besides their utility for monitoring Cre-mediated recombination, we show that in IRG mice red and green native fluorescence can be imaged simultaneously in thick tissue sections by confocal microscopy allowing for complex reconstructions to be created that are suitable for analysis of neuronal morphologies as well as neurovascular interactions in brain. IRG mice should provide a versatile tool for analyzing complex cellular relationships in both neural and nonneural tissues.