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At least 19 recordsLinked to original sources
PubMed2001

Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.

BACKGROUND: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize beta-gal in living tissue. RESULTS: In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (beta-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. CONCLUSIONS: In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues.

Animals↗
PubMed2007

Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies.

The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a "universal Cre-deleter" strain and with mice expressing Cre recombinase in a T lineage-specific manner. Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding "leaky" reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny.

Animals↗
PubMed2006

Cre reporter system to monitor the translocation of type III secreted proteins into host cells.

Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

Animals↗
PubMed2001

Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells.

The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescent protein is produced. Testing this system in transiently transfected cells showed that it produced robust signals (90% of the cells converted from red to green) when equal amounts of the plasmids encoding Cre recombinase and the Cre Stoplight were used. A 1:100 ratio of enzyme to reporter plasmid produced similar results, and a 1:10000 ratio was necessary to significantly reduce the number of cells converting to green (1%).

Cell Line↗
PubMed2000

Characterisation of human melatonin mt(1) and MT(2) receptors by CRE-luciferase reporter assay.

A cyclic AMP response element (CRE)-luciferase reporter gene assay was used to characterise the functional responses of human melatonin mt(1) and human melatonin MT(2) receptors, stably expressed in the human embryonic kidney cell line HEK293, to a series of six naphthalenic analogues of melatonin. By comparison to the observed melatonin-mediated inhibition of stimulated luciferase levels the naphthalenic series was identified as comprising agonists, partial agonists and one antagonist of melatonin mt(1) and melatonin MT(2) receptor function. Three of the agonist/partial agonist members of this series were also identified as displaying a functional selectivity for the melatonin MT(2) receptor. Competitive displacement of 2-[125I]iodomelatonin binding to the ovine pars tuberalis melatonin ML(1) receptor demonstrated a close correlation to the observed functional luciferase responses of the human melatonin mt(1) receptor. We conclude that the CRE-luciferase reporter gene assay provides an effective functional screening method for the pharmacological characterisation of human melatonin receptor subtypes.

Acetamides↗
PubMed2004

Cytokines increase CRE binding but decrease CRE-mediated reporter activity in rat hepatocytes by increasing c-Jun.

The cyclic AMP response element (CRE) has been implicated in the regulation of the expression of many genes and cellular processes important in hepatocyte function. CRE sites exist in the promoter regions of several genes expressed during inflammation. Numerous studies on the role of CRE in hepatocyte gene expression have been performed in resting hepatocytes, but the role of CRE during inflammation is unknown. To evaluate the regulation of CRE-mediated transcription during sepsis, cultured hepatocytes were exposed to proinflammatory cytokines and lipopolysaccharide (LPS) was injected into rats. Nuclear proteins were collected and CRE binding activity measured by electromobility shift assay (EMSA) using a consensus CRE oligonucleotide. CRE binding activity was increased in vitro by cytokines and in vivo by LPS administration but CRE-dependent reporter activity was decreased by cytokine stimulation. A c-jun N-terminal kinase (JNK) inhibitor reversed the cytokine-induced increase in CRE binding and increased CRE-dependent reporter activity. Supershift assays indicated that cyclic AMP response element binding protein (CREB) and c-Jun proteins were included in the CRE binding complex. CREB induced and c-Jun suppressed reporter activity using a CRE-dependent construct transfected into cultured primary hepatocytes. In conclusion, these data demonstrate that proinflammatory cytokines regulate CRE binding and activity in cultured hepatocytes and suggest that sepsis-induced changes in CRE binding may participate in the cellular response to inflammation.

Animals↗
PubMed2005

Cellular inheritance of a Cre-activated reporter gene to determine Paneth cell longevity in the murine small intestine.

Here, we exploit an absolute differential between stem and progeny cells in their ability to express Cre from a somatically inducible transgene to determine the longevity of intestinal Paneth cells. In the Ahcre transgenic line induction of Cre recombinase allows constitutive activation of a Cre-activated reporter in intestinal precursors but not in Paneth cells. The time taken for Paneth cells to inherit the reporter (EYFP) was measured in adult Ahcre/R26R-EYFP animals. Using confocal microscopy of TOPRO-3-stained sections, both precursors and Paneth cells were identified and subsequently scored for EYFP expression. It takes up to 57 days for Paneth cells to inherit the reporter, making them three times longer-lived than previously indicated using nucleotide incorporation and suggesting that such determinations of cell turnover may be significant underestimates.

Animals↗
PubMed2002

The effect of ICER on screening methods involving CRE-mediated reporter gene expression.

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.

Animals↗
PubMed2006

GFAP-positive progenitor cells produce neurons and oligodendrocytes throughout the CNS.

Once thought to merely act as scaffolds in neuronal migration, recent evidence suggests that radial glia may serve as progenitors for the majority of neurons in the CNS. Cre/loxP fate-mapping experiments were carried out using a fragment of a glial-specific promoter (glial fibrillary acidic protein; GFAP) to drive expression of Cre recombinase. We show that GFAP+ progenitor cells give rise to neurons and oligodendrocytes throughout the CNS. We find very little regional heterogeneity in the neurogenic potential of radial glia between dorsal and ventral telencephalon. Additionally, radial glia serve as precursors for subpopulations of interneurons in the ventral telencephalon. Interestingly, the human GFAP promoter but not the mouse GFAP promoter is active in oligodendrocyte progenitor cells. We also demonstrate that the most commonly used Cre reporter lines are very inefficient in detecting Cre-dependent recombination in astrocytes and describe a new Cre reporter line for assessing recombination in astrocytes.

Animals↗
PubMed2004

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.

Animals↗
PubMed2007

Conditional mutagenesis by cell-permeable proteins: potential, limitations and prospects.

The combination of two powerful technologies, the Cre/loxP recombination system and the protein transduction technique, holds great promise for the advancement of biomedical and genome research by enabling precise and rapid control over mutation events. Protein transduction is a recently developed technology to deliver biologically active proteins directly into mammalian cells. It involves the generation of fusion proteins consisting of the cargo molecule to be delivered and a so-called protein transduction domain. Recently, the derivation of cell permeable variants of the DNA recombinase Cre has been reported. Cre is a site-specific recombinase that recognizes 34 base pair loxP sites and has been widely used to genetically engineer mammalian cells in vitro and in vivo. Recombinant cell-permeable Cre recombinase was found to efficiently induce recombination of loxP-modified alleles in various mammalian cell lines. Here we review recent advances in conditional expression and mutagenesis employing cell-permeable Cre proteins. Moreover, this review summarizes recent findings of studies aimed at deciphering the molecular mechanism of the cellular uptake of cell-permeable fusion proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.

The Cre/loxP system has become an important tool in designing postintegrational switch mechanisms for transgenes in mice. The power and spectrum of application of this system depends on transgenic mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. We have developed a novel mouse line that acts as a Cre reporter. The mice, designated Z/EG (lacZ/EGFP), express lacZ throughout embryonic development and adult stages. Cre excision, however, removes the lacZ gene, which activates expression of the second reporter, enhanced green fluorescent protein. We have found that the double-reporter Z/EG line is able to indicate the occurrence of Cre excision from early embryonic to adult lineages. The advantage of the Z/EG line is that Cre-mediated excision can be monitored in live samples and that live cells with Cre-mediated excision can be isolated using a single-step FACS. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

Alkaline Phosphatase↗
PubMed2004

Impaired CREB-1 phosphorylation in antifolate-resistant cell lines with down-regulation of the reduced folate carrier gene.

The human reduced folate carrier (hRFC) is the dominant transporter for the uptake of antifolates used in cancer chemotherapy. We have shown recently that decreased cAMP-responsive element (CRE)-dependent transcription contributes to the loss of hRFC gene expression in multiple antifolate-resistant cell lines. This was associated with markedly decreased levels of phosphorylated cAMP response element-binding protein 1 (pCREB-1) and CRE-binding. Consistent with the autoregulation of CREB-1 gene expression by pCREB-1, prominently decreased CREB-1 mRNA levels were observed in antifolate-resistant cells. We therefore explored the possibility that these cells were defective in CREB-1 phosphorylation, thereby resulting in down-regulation of some cAMP-responsive genes. Two-dimensional gel electrophoresis revealed that CREB-1 and its phosphoisoforms were markedly decreased in these cells. Treatment with forskolin, an activator of adenylyl cyclase, restored both CREB-1 and pCREB-1 levels; this resulted in the restoration of CRE-binding, CRE-reporter activity, and CREB-1 and RFC mRNA levels. Hence, the protein kinase A pathway was examined using various agents that augment intracellular cAMP levels, including cholera toxin, an upstream agonist that renders stimulatory G-proteins (Galphas) constitutively active. Treatment of antifolate-resistant cells with these agents resulted in the restoration of pCREB-1 levels and CRE-reporter activity. Furthermore, transient transfection with a constitutively transcriptionally active VP16-CREB-1 that does not require phosphorylation for its activity resulted in restoration of CREB mRNA levels but not pCREB-1 levels. This is the first demonstration that resistance to various antifolates may potentially be associated with impaired activity of Galphas or their coupled receptors, resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes.

1-Methyl-3-isobutylxanthine↗
PubMed2006

Generation and maintenance of Dmbx1 gene-targeted mutant alleles.

Dmbx1 encodes a paired-like homeodomain protein that is expressed in neural tissues at mouse embryonic and postnatal stages. We previously generated two Dmbx1 mutant alleles, Dmbx1 (-) and Dmbx1 ( z ), by homologous recombination in mouse embryonic stem (ES) cells. In this article we report the generation of three novel Dmbx1 mutant alleles, Dmbx1 (tauZ ), Dmbx1 (tauG ), and Dmbx1 ( Cre ), that carry the intronic insertion of tau (tau)-lacZ, tau-eGFP, and Cre reporter genes, respectively. Dmbx1 (tauZ ) and Dmbx1 (tauG ) recapitulated the Dmbx1 expression, and the reporter gene expression was detected in the diencephalon and mesencephalon during embryogenesis. The crossing of Dmbx1 ( Cre ) mice with Rosa26 reporter mice identified the Cre-mediated DNA excision in the postnatal midbrain, cerebellum, medulla oblongata, and spinal cord. To maintain the Dmbx1 mutant alleles without genotyping, we crossed Dmbx1 mutant mice with Inv4(1) ( Brd ) mice that possess the inversion between D4Mit117 and D4Mit281 on Chromosome 4, where Dmbx1 is located. The intercrossing of the non-agouti (a/a) albino (Tyr ( c-Brd )/Tyr ( c-Brd )) Dmbx1 mutant mice carrying Inv4(1) ( Brd ) tagged with K14-Agouti and Tyrosinase coat-color markers resulted in the generation of dark brown Dmbx1 wild-type [Inv4(1) ( Brd )/Inv4(1) ( Brd )], light brown Dmbx1 heterozygous [Dmbx1 ( tm )/Inv4(1) ( Brd )], and albino Dmbx1 homozygous (Dmbx1 ( tm )/Dmbx1 ( tm )) mutant mice. To our knowledge, this is the first demonstration of the proof-of-principle of the maintenance of viable gene-targeted alleles using coat-color-tagged nonlethal balancer chromosomes.

Alleles↗
PubMed1995

Analysis of perinatal gene expression: hormone response elements mediate activation of a lacZ reporter gene in liver of transgenic mice.

The transcription of genes encoding gluconeogenic enzymes is tightly regulated during the perinatal period. These genes are induced by glucagon (cAMP) and glucocorticoids and repressed by insulin. To address the role of cAMP and glucocorticoids in the physiological activation of genes encoding gluconeogenic enzymes in the perinatal period, transgenic mice have been generated with chimeric constructs containing the reporter gene lacZ under the control of hormone response elements. The activity of the transgene is restricted to the liver by the presence of the enhancers from the alpha-fetoprotein gene and its transcription is driven by a promoter that contains a TATA box linked to either cAMP response elements (CREs) or glucocorticoid response elements (GREs). We demonstrate cAMP and glucocorticoid regulation, liver-specific expression, and perinatal activation of the reporter gene. These data indicate that the CRE and GRE are, independently, necessary and sufficient to mediate perinatal gene activation. Perinatal activation was not impaired when a CRE reporter transgene was assayed in mice that contain a targeted mutation of the CRE-binding protein (CREB) gene, providing further evidence for functional redundancy among the members of the CREB/ATF gene family.

Animals↗