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PubMed2004

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells.

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.

Animals↗

PubMed2004

Cre recombinase specificity defined by the tau locus.

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.

Animals↗

PubMed2005

Cre-mediated recombination in cell lineages that express the progesterone receptor.

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.

Animals↗

PubMed2005

Transgenic mice that express Cre recombinase in hypertrophic chondrocytes.

In order to investigate the physiological control of hypertrophic chondrocytes which present the terminally differentiated form of chondrocytes, we generated a mouse line expressing the Cre recombinase under the control of the mouse type X collagen (Col10a1) promoter. In situ hybridization analysis demonstrated the expression of Col10a1-Cre transgene in hypertrophic chondrocytes of femur at postnatal day 2 (P2). In order to test the excision activity of the Cre recombinase, the Col10a1-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4co/co) and the reporter line ROSA26. Multiple tissue PCR of Col10a1-Cre;Smad4co/+ mice revealed the restricted Cre activity in tissues containing hypertrophic chondrocytes. LacZ staining revealed that the Cre activity was observed in the cartilage primordia of ribs at E14.5 and only detected in the lower hypertrophic region of ribs at P1. These data suggest that the Col10a1-Cre mouse line described here could be used to achieve conditional gene targeting in hypertrophic chondrocytes.

Animals↗

PubMed2005

Generation and characterization of the PKC gamma-Cre mouse line.

PKCgamma is a protein kinase C (PKC) isoform that is expressed in the central nervous system (CNS). We generated a PKCgamma-Cre mouse line and characterized the expression and activity of the Cre protein. Our studies show that Cre expression largely recapitulates the endogenous expression of PKCgamma. Several major sites of Cre expression are the cerebral cortex, hippocampus, thalamus, amygdala, and spinal cord. Examination of PKCgamma-Cre/ROSA26 mice reveals a similar X-gal staining pattern in the CNS, indicating that Cre recombinase is capable of removing LoxP sites in vivo. These data indicate that the PKCgamma-Cre mouse line could be a useful reagent for generating Cre-mediated tissue-specific knockouts in the CNS.

Animals↗

PubMed2006

Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.

Cre-mediated conditional gene targeting has been shown to be successful in many cell and tissue types. However, gene recombination in the uterus with heterogeneous cell types by Cre activation is not yet well established. Using recombinant adenoviruses expressing a functional Cre (ADV-Cre) and ROSA26 reporter mice, we show here that ADV-Cre infused intraluminally in a small volume (10 microl) conditionally excises the loxP site, resulting in lacZ expression in uterine luminal epithelial cells without significantly affecting pregnancy. In contrast, a similar intraluminal infusion of ADV-Cre in a larger volume (50 microl) damages the normal architecture and integrity of the luminal epithelium, inducing gene recombination in the underneath stromal cells, with disruption of pregnancy. Further, decidualizing stromal cells at the implantation sites can be targeted by ADV-Cre after intravenous administration on days 5-6. This route of administration also elicits Cre activity in other tissues, including the liver, spleen, ovary, and, more remarkably, in the adrenal cortex. These findings demonstrate the feasibility of achieving conditional expression or deletion of specific genes in uterine cells at desired times and physiological states.

Adenoviridae↗

PubMed2003

Transgenic mice with hematopoietic and lymphoid specific expression of Cre.

Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants. We have generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R-EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single-cell level. Analysis showed that the vav promoter elements were able to direct Cre-mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre-mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre-transgenic lines will be useful in generating tissue-specific gene deletions within all the cells of hematopoietic or lymphoid tissues.

Animals↗

PubMed1999

Carbon regulation of ribosomal genes in Neurospora crassa occurs by a mechanism which does not require Cre-1, the homologue of the Aspergillus carbon catabolite repressor, CreA.

Transcription of the ribosomal protein and 40S rRNA genes is coordinately regulated during steady state growth and carbon shifts in Neurospora crassa. Recognition sequences for the Aspergillus nidulans carbon catabolite repressor, CreA, overlap transcriptional elements of a 40S rRNA gene and the crp-2 ribosomal protein gene. They also occur in similar locations in the promoters of several other ribosomal protein genes. Substitutions encompassing the -74 and -167 CreA consensus sequences in the crp-2 promoter result in a decrease in transcription. A cDNA encoding the N. crassa homologue of CreA was cloned and designated Cre-1. The Cre-1 protein is 45% identical to CreA from A. nidulans. Cre-1 protein produced in Escherichia coli binds to the CreA sites in the promoters of the 40S rRNA and crp-2 genes. An amino acid change from histidine (92) to threonine changed the Cre-1 binding specificity from (5'G/CC/TGGG/AG3') to (5'G/CC/TGGCG3'). Base substitutions in the Cre-1 binding sites of the crp-2 promoter disrupted binding of wildtype Cre-1 in vitro but had no effect on transcription during steady state growth or carbon shifts, indicating that regulation of ribosomal genes by carbon source is not mediated by Cre-1, but via different proteins binding the Cre-1 sites and the Dde boxes.

Amino Acid Sequence↗

PubMed1988

Properties of a mutant Cre protein that alters the topological linkage of recombination products.

The bacteriophage P1 Cre-loxP site-specific recombination system consists of two components: the Cre recombinase protein and the loxP DNA sequence where recombination takes place. We report here on the analysis of a mutation in the cre structural gene that produces a mutant protein with altered recombination properties. The mutant protein, Cre111, carries out recombination at a much slower rate than the wild-type Cre protein. To determine why the reaction is slow, we have examined a number of activities associated with Cre-mediated recombination. Our results indicate that the binding of Cre111 to the loxP site is comparable to wild-type Cre. Furthermore, the rate at which Cre111 resolves Holliday structures, an intermediate in this recombination reaction, is also comparable to wild-type Cre. Thus, DNA binding and resolution of the intermediate are not affected, suggesting that either synapsis, the process of bringing two lox sites together, or the first strand exchange event, could be affected in the mutant protein. The types of DNA products formed following recombination of a supercoiled substrate can reflect mechanisms of synapsis. Wild-type Cre generates mainly topologically unlinked and unknotted circular products. This suggests that the wild-type protein brings two lox sites together in a way that excludes the entanglement of supercoils present in the substrate DNA. In contrast, when Cre111 recombines a supercoiled molecule it generates many complicated catenanes and knotted DNA products. Presumably, the supercoils present in the DNA substrate are being trapped in the reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

Coliphages↗

PubMed2000

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.

Alleles↗

PubMed1997

Transient CRE- and kappa B site-binding is cross-regulated by cAMP-dependent protein kinase and a protein phosphatase in mouse splenocytes.

Cyclic AMP regulates a variety of cellular responses through activation of cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA, in turn, activate cAMP responsive element (CRE) and nuclear factor-kappa B (NF-kappa B) binding proteins. In this study, we demonstrated that binding activity to both CRE and kappa B sites in nuclear extracts from spleen cells is modulated by PKA in a time-dependent manner. Electrophoretic mobility shift assays showed that binding by transcription factors to either the CRE or kappa B motif was rapidly up-regulated by cAMP, with maximum binding detected at 30 min in response to forskolin stimulation of splenocytes. This was followed by a steady decline in CRE and kappa B thereafter reaching basal levels by 2 hr. This up-regulation in CRE and kappa B binding was closely associated with an enhancement of PKA activity which was maximum at 30 min following forskolin stimulation. However, unlike the binding of regulatory factors to CRE and kappa B motifs which was very transient, peak PKA activity was sustained for 2 hr. Interestingly, okadaic acid, a protein phosphatase inhibitor, prevented the decline in protein binding to CRE and kappa B motifs 2 hr following forskolin stimulation and actually produced a slight increase at 30 min. These data suggest that binding by transcription factors to CRE and kappa B sites are up-regulated concomitantly with PKA activation but subsequently down-regulated by a protein phosphatase.

Animals↗

PubMed1998

Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase.

Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes. Unfortunately, there are few means available to measure Cre protein expression in vivo. We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989. J. Virol. 63, 2325-2334). The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody. The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites. We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse. In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.

3T3 Cells↗

PubMed2001

Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.

To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.

Alleles↗

PubMed2001

Stage-and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice.

To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.

Animals↗

PubMed2002

Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes.

Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.

Cells, Cultured↗

PubMed1999

Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.

Effective use of conditional Cre recombinase-loxP gene modification requires Cre-expressing mouse strains with defined patterns of expression. To assess the in vivo functionality of Cre-expressing mice, we have engineered an improved reporter strain for monitoring Cre-mediated excisions. The beta-galactosidase-neomycin phosphotransferase fusion gene (betageo)-trapped ROSA26 locus was modified by gene targeting such that betageo is expressed only after Cre-mediated excision of loxP-flanked DNA sequences. betageo from the excised ROSA26 allele is expressed ubiquitously in embryos and adult mice. By mating the reporter strain with Cre-expressing transgenic mice, we have shown that the loxP-flanked ROSA26 allele is accessible to Cre during early embryogenesis, as well as in a specific hematopoietic lineage (T lymphocytes). This improved reporter strain should facilitate monitoring in vivo Cre-mediated excision events in a variety of experimental contexts.

Animals↗

PubMed2000

Trans complementation of variant Cre proteins for defects in cleavage and synapsis.

The Cre recombinase is a member of the integrase family of conservative site-specific recombinases. These proteins share five conserved catalytic residues, one of which is a tyrosine that acts as the nucleophile to attack the scissile phosphodiester bond in the DNA target. Recombination by the Cre recombinase takes place in a supramolecular structure called a synapse that consists of four molecules of Cre bound to two DNA target sequences called lox sites. The synapse is held together by an intricate network of protein-protein interactions. They bend the two sites into square planar structure that resembles a Holliday intermediate. We have studied three mutant Cre proteins that appear to have defects in synapsis (Cre A36V, Cre T41F, and Cre G314R). We found that they were unable to carry out strand cleavage but that cleavage occurred if they were mixed with a cleavage-defective Cre protein that lacks the catalytic nucleophilic tyrosine residue. The three variant proteins could also be complemented for the formation of a novel structure ("complexV"), which may be a cleaved synaptic intermediate. We suggest that these three mutant proteins have a defect in DNA bending and discuss the relationship between bending, synapsis, and cleavage.

DNA↗

PubMed2003

Cre induces an asymmetric DNA bend in its target loxP site.

Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.

Amino Acid Substitution↗