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.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Generation of Frizzled10-Cre transgenic mouse line: a useful tool for the study of dorsal telencephalic development

Wnt signaling plays an important role in regulating cortical and hippocampal development, but many of the other molecular mechanisms underlying dorsal telencephalic development are largely unknown. We are taking advantage of the highly regionalized expression patterns of signaling components of the Wnt pathway to generate new mouse lines that will be useful for studying forebrain development. Here, we describe a transgenic mouse line where Cre is driven by the promoter of the Wnt receptor, Frizzled10. In these mice, Cre activity is mainly detected in the dorsal telencephalon during development and is confined to the pyramidal cell fields in the adult hippocampus. The Cre recombinase has very high efficiency when assayed by crossing the transgenic line with the ROSA26 reporter line. Thus, this Cre line will be useful for the study of dorsal telencephalic development and conditional inactivation of target genes in the cortex and hippocampus.     

Tamoxifen-inducible NaV1.8-CreERT2 recombinase activity in nociceptive neurons of dorsal root ganglia

To explore the function of genes expressed in adult mouse nociceptive neurons, we generated heterozygous knock-in mice expressing the tamoxifen-inducible Cre recombinase construct CreERT2 downstream of the Na(V)1.8 promoter. CreERT2 encodes a Cre recombinase (Cre) fused to a mutant estrogen ligand-binding domain (ERT2) that requires the presence of tamoxifen for activity. We have previously shown that heterozygous Na(V)1.8-Cre mice will delete loxP flanked genes specifically in nociceptive sensory neurons from embryonic day 14. We therefore used the same strategy of homologous recombination and mouse generation, substituting the Cre cassette with CreERT2. No functional Cre recombinase activity was found in CreERT2 mice crossed with reporter mice in the absence of tamoxifen. We found that, as with Na(V)1.8-Cre mice, functional Cre recombinase was present in nociceptive sensory neurons after tamoxifen induction in vivo. However, the percentage of dorsal root ganglion (DRG) neurons expressing functional Cre activity was much reduced (<10% of the number found in the Na(V)1.8-Cre mouse). We also examined Cre recombinase activity in sensory neurons in culture. After treatment with 1 muM tamoxifen for 48 h, 15% of DRG neurons showed Cre activity. Na(V)1.8-CreERT2 animals may thus be useful for single cell studies of the functional consequences of gene ablation in culture, but are unlikely to be useful for behavioral studies.     

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.     

Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia

The use of Cre-loxP technology for conditional mutagenesis in pain pathways had been restricted by the unavailability of mice expressing Cre recombinase selectively in functionally distinct components of the nociceptive system. Here we describe the generation of transgenic mouse lines which express Cre recombinase selectively in sensory ganglia using promoter elements of the Na(v)1.8 gene (Scn10a). Cre-mediated recombination was greatly evident in all nociceptive and thermoreceptive neurons of the dorsal root ganglia and trigeminal ganglia, but only in a small proportion of proprioceptive neurons. Cre-mediated recombination was not detectable in the brain, spinal cord, or any nonneural tissues and began perinatally after invasion of primary afferents into the developing spinal cord. Thus, these mice enable selective deletion of genes in subsets of sensory neurons and offer a wide scope for studying potential functions of genes in pain perception, independent of secondary effects arising from developmental defects or global gene ablation.     

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.     

Analysis of dopamine transporter gene expression pattern -- generation of DAT-iCre transgenic mice

The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon-improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome-dopamine transporter-iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome-dopamine transporter-iCre transgene and a construct expressing the beta-galactosidase gene after Cre-mediated recombination. In situ studies showed that beta-galactosidase (5-bromo-4-chloroindol-3-yl beta-D-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of beta-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome-dopamine transporter-iCre transgenic line is a unique tool for targeting Cre/loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.     

A transgenic mouse line expressing cre recombinase in pancreatic β-cells

Transgenic mouse lines expressing Cre recombinase in a cell-specific and tissue-specific manner are essential tools for studying gene function and for developing suitable models for human diseases. Here, we used an expression cassette containing the full 5' untranslated region of the porcine insulin gene to generate a mouse line expressing Cre recombinase specifically in pancreatic β-cells by pronuclear DNA microinjection. We obtained a founder animal that transmitted the construct to its descendants in a Mendelian fashion and whose descendants showed a clear activation of β-galactosidase expression in pancreatic β-cells after crossing into the ROSA26 lacZ reporter mouse line. Cre expression in other organs was negative except for the kidney, intestine, and the cerebral pons where β-galactosidase activity was detected in a small percentage of the cells. This new mouse line is a valuable tool for recombination of floxed alleles in pancreatic β-cells in vivo.     

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.     

Extracellular Vesicle-Mediated Transfer of Genetic Information between the Hematopoietic System and the Brain in Response to Inflammation

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.     

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。     

DARPP-32 genomic fragments drive Cre expression in postnatal striatum

To direct Cre-mediated recombination to differentiated medium-size spiny neurons (MSNs) of the striatum, we generated transgenic mice that express Cre recombinase under the regulation of DARPP-32 genomic fragments. In this reported line, recombination of an R26R reporter allele occurred postnatally in the majority of medium-size spiny neurons of the dorsal and ventral striatum (caudate nucleus and nucleus accumbens/olfactory tubercle), as well as in the piriform cortex and choroid plexus. Although regulatory fragments were selected to target MSNs, low levels of Cre-recombinase expression, as detected by beta-galactosidase activity from the R26R reporter gene, were also apparent in widely dispersed areas or cells of the forebrain and hindbrain. These included the primary and secondary motor cortex, and association cortex, as well as in the olfactory bulb and cerebellar Purkinje cells. Notably, expression in these regions was well below that of endogenous DARPP-32. Analysis of colocalization of beta-galactosidase, as detected either by histochemistry or immunocytochemistry, and DARPP-32 revealed double-labeling in almost all DARPP-32-expressing MSNs in the postnatal striatum, but not in extrastriatal regions. The DARPP-32Cre transgenic mouse line thus provides a useful tool to specifically express and/or inactivate genes in mature MSNs of the striatum.     

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreER(TM)) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis.     

Cerebellar granule cell Cre recombinase expression

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.     

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid.To study the function of gastric parietal cells during gastric epithelium homeostasis,we generated a transgenie mouse line,namely,Atp4b-Cre,in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H+-,K+-ATPase gene(Atp4b).In order to test the tissue distribution and excision activity of Cre recombinase in vivo,the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles(Smad4Co/Co).Multiple-tissue PCR of Atp4b-Cre;Smad4Co/+mice revealed that the recombination only happened in the stomach.As indicated by LacZ staining,ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells.These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.     

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.     

Efficient bicistronic expression of cre in mammalian cells

Cre recombinase-mediated DNA recombination is proving to be a powerful technique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalian cells and have tested its efficiency of expression in a bicistronic message. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either of two introns resulted in an approximately 2-fold increase in recombination frequency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus internal ribosome entry site (IRES). Recombination frequencies were only reduced 2-fold relative to a comparable monocistronic cre gene. The latter results indicate that it will be possible to generate transgenic mouse strains having tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.