Podocyte-specific expression of cre recombinase in transgenic mice

We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.     

Generation of a mouse line expressing Cre recombinase in glycinergic interneurons

In caudal regions of the CNS, glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic glycine transporter 2 (GlyT2) promoter fragment. Mating of GlyT2‐Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as glycinergic interneurons by staining with GlyT2‐ and glycine‐immunoreactive antibodies; here, >80% of the glycine‐immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2‐Cre mice are a useful tool for the genetic manipulation of glycinergic interneurons. genesis 48:437–445, 2010. © 2010 Wiley‐Liss, Inc.     

Selection of primary cell cultures with cre recombinase induced somatic mutations from transgenic mice.

Deletion of genes in defined cell types has been achieved using a combination of gene targeting techniques and the Cre- lox P recombination system. Here we present a method to selectively isolate genetically altered primary cell cultures based on the permanent activation of a drug-resistance gene by the Cre recombinase. Transgenic mice were generated harboring a dormant form of the hygromycin resistance gene. This mouse line was crossed with mice carrying a constitutive Cre gene and an endogenous floxed allele. Primary fibroblasts established from triple transgenic embryos displayed not only hygromycin resistance but also recombination of the endogenous floxed allele. These results prove the potential of this approach.     

Meox1Cre: a mouse line expressing Cre recombinase in somitic mesoderm

We developed a novel strategy based on in vitro DNA transposition of phage Mu to construct vectors for "knock-in" of the gene encoding Cre recombinase into endogenous loci in embryonic stem cells. This strategy was used to introduce Cre into the mouse Meox1 locus, which was expected to drive Cre expression in the presomitic and somitic mesoderm. In embryos heterozygous for both Meox1(Cre) and R26R or Z/AP reporter alleles, specific and efficient recombination of the reporter alleles was detected in the maturing somites and their derivatives, including developing vertebrae, skeletal muscle, back dermis, as well as endothelium of the blood vessels invading the spinal cord and developing limbs. In contrast to the somitic mesoderm, Cre activity was not observed in the cranial paraxial mesoderm. Thus, the Meox1(Cre) allele allows detailed fate-mapping of Meox1-expressing tissues, including derivatives of the somitic mesoderm. We used it to demonstrate dynamic changes in the composition of the mesenchyme surrounding the developing inner ear. Meox1(Cre) may also be used for tissue-specific mutagenesis in the somitic mesoderm and its derivatives.     

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

Microinjection of cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes.

We have developed a strategy for producing single copy transgenic mouse lines using Cre-loxP site specific recombination. The method is based on transient expression of the recombinase after injection of in vitro transcribed mRNA into the cytoplasm of fertilised eggs containing multiple copies of the transgene. The success rate of the recombination event is 100% (15 out of 15).     

Neuron-specific enolase-cre mouse line with cre activity in specific neuronal populations

To establish genetic tools for conditional gene deletion in mouse neurons, we generated two independent neuron-specific enolase (Nse)-cre transgenic lines. The transgenic line termed Nse-cre(CK1) showed cre activity in most neuronal regions in the nervous system, while the Nse-cre(CK2) line exhibited a unique cre activity that has not been reported in other cre transgenic lines. Nse-cre(CK2) cre activity was detectable from embryogenesis and mostly restricted to neuronal regions. In postnatal brain, the Nse-cre(CK2) line exhibited cre activity limited to differentiated neurons in the cerebral cortex and hippocampus. Cre activity was assayed in several internal organs and sporadic activity was limited to the kidney and testis. We conclude that these cre lines will be useful for studying loss of gene function in specific neuronal populations.     

Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth

Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging. In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter. The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts. The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice. The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth. The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development. Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age. We could not detect Cre recombinase activity in any other cells, including ameloblasts. These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis.     

A transgenic mouse strain expressing four drug-selectable marker genes.

Murine embryonic stem (ES) cells are commonly cultured on feeder layers of primary murine embryonic fibroblasts (MEFs). Because gene targeting experiments often involve sequential selection for multiple-drug resistance in single ES cell lines, we have developed a new mouse strain which represents an economical donor for the production of multiple-drug resistant MEFs. MEFs prepared from the DR-4 mouse strain displayed resistance to concentrations of the drugs G418, 6-thioguanine, puromycin and hygromycin well above those used normally for the selection of drug-resistant ES cells.     

A Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka

Cre/loxP-dependent expression of fluorescent proteins represents a powerful biological tool for cell lineage, fate-mapping, and genetic analysis. Live tissue imaging has significantly improved with the development of far-red fluorescent proteins, with optimized spectral characteristics for in vivo applications. Here, we report the generation of the first transgenic mouse line expressing the far-red fluorescent protein Katushka, driven by the hybrid CAG promoter upon Cre-mediated recombination. After germ line or tissue-specific Cre-driven reporter activation, Katushka expression is strong and ubiquitous, without toxic effects, allowing fluorescence detection in fresh and fixed samples from all tissues examined. Moreover, fluorescence can be detected by in vivo noninvasive whole-body imaging when Katuhska is expressed exclusively in a specific cell population deep within the animal body such as pancreatic beta cells. Thus, this reporter model enables early, widespread, and sensitive in vivo detection of Cre activity and should provide a versatile tool for a wide spectrum of fluorescence and live-imaging applications.     

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.     

A transgenic mouse expressing human CYP4B1 in the liver

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.     

Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancer

Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0-13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5-E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines.     

他莫昔芬诱导的Cre重组酶在hGfapCreERT2转基因鼠小脑中的表达研究

目的探讨他莫昔芬诱导的hGfapCreERT2转基因鼠小脑中表达Cre重组酶的细胞类型。方法 hGfapCre-ERT2/Rosa26R转基因小鼠在胚胎晚期和出生早期用他莫昔芬诱导Cre重组酶表达,对小脑组织切片行X-gal染色,然后用细胞种类特异性抗体进行免疫组织化学染色,并和X-gal染色双重标记。结果在出生后第7天(P7)、第14天(P14)和第60天(P60),X-gal阳性染色和胶质细胞抗体Blbp阳性染色共标记,和神经元抗体Neun、浦肯野细胞抗体Calbindin及少突胶质细胞前体细胞抗体NG2不共标。结论自胚胎晚期第17.5天(E17.5)后用他莫昔芬诱导hGfapCreERT2转基因鼠,发现Cre重组酶特异性在小脑星形胶质细胞中表达,不在神经元、浦肯野细胞、少突胶质细胞前体细胞中表达。