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PubMed1997

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor gamma (TCR V gamma) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (V gamma LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted V gamma locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (V gamma delta) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the V gamma delta locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined V gamma LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the V gamma LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes.

Animals↗

PubMed2005

Cre-mediated site-specific recombination in zebrafish embryos.

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.

Animals↗

PubMed2002

Codon-improved Cre recombinase (iCre) expression in the mouse.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Animals↗

PubMed2005

MyoD-cre transgenic mice: a model for conditional mutagenesis and lineage tracing of skeletal muscle.

The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters in vivo.

Animals↗

PubMed2005

Transgenic expression of Cre recombinase in mitral/tufted cells of the olfactory bulb.

Olfactory information is conveyed from the periphery to the olfactory cortices through mitral and tufted (M/T) cells in the olfactory bulb. A mouse with a specific expression of Cre recombinase in M/T cells is essential for genetic marking of M/T cells and manipulating their properties. Protocadherin 21 (Pcdh21) expression is highly restricted to M/T cells. Here we report a transgenic mouse line, Pcdh21-Cre, in which 10-kb mouse Pcdh21 promoter drives the expression of Cre recombinase. In Pcdh21-Cre mice, Cre recombinase activity is predominantly detected in M/T cells, visualized with the anti-CFP immunostaining in offspring of a cross between Pcdh21-Cre and the reporter Rosa26-loxP-stop-loxP-CFP strain. These results demonstrate that the 10-kb Pcdh21 promoter can drive transcription in M/T cells and Pcdh21-Cre mice can be used to excise floxed DNA fragments in M/T cells, which provides a valuable tool to reveal the structure and function of the central olfactory circuits.

Animals↗

PubMed2005

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.

Animals↗

PubMed2003

Regulation of CRE-mediated transcription in mouse brain by amphetamine.

Previous work has demonstrated that acute and chronic administration of amphetamine causes phosphorylation of the transcription factor CREB, the cAMP response element (CRE) binding protein, in striatum, a brain region important for the behavioral actions of the drug. To determine whether such phosphorylation is associated with changes in CREB transcriptional activity, we mapped beta-galactosidase (beta-gal) expression in a CRE-LacZ transgenic mouse, in which the beta-gal reporter is downstream of CRE sequences, following acute or chronic amphetamine administration. We found that acute amphetamine induced beta-gal expression in a relatively small number of brain regions, including nucleus accumbens (ventral striatum), amygdala, ventral tegmental area, and locus coeruleus. Chronic amphetamine generally produced greater changes in CRE-mediated transcription in most brain regions and induced CRE-transcription in several regions unaffected by acute drug exposure. Interestingly, amphetamine regulation of CRE activity differed dramatically between males and females. In nucleus accumbens, beta-gal expression colocalized predominantly with dynorphinergic neurons after acute amphetamine administration, while chronic administration induced beta-gal expression in both dynorphinergic and enkephalinergic neurons. In ventral tegmental area, acute and chronic amphetamine induced beta-gal expression mainly in dopaminergic neurons, while induction in the locus coeruleus occurred mainly in nonnoradrenergic neurons. This study identifies several brain regions where CRE-mediated transcription may play a key role in the development of neuronal plasticity associated with amphetamine administration.

Amphetamine↗

PubMed2001

Reduction in cyclin D1/Cdk4/retinoblastoma protein signaling by CRE-decoy oligonucleotide.

We have previously demonstrated that the activation of p53 signaling may contribute to tumor growth inhibition by the CRE-decoy oligonucleotide containing CRE sequence (5'-TGACGTCA-3') (Lee et al., Biochemistry 39, 4863-4868, 2000). However, growth inhibition by CRE-decoy treatment was also observed in tumor cells containing a mutant p53 (Park et al., J. Biol. Chem. 274, 1573-1580, 1999). To understand additional mechanisms of the decoy oligonucleotide, we investigated the effect on cyclin D1 expression and a cyclin D1/Cdk4/retinoblastoma protein (pRB) signaling pathway. Here we show that in MCF7 breast cancer cells the CRE-decoy competed with cyclin D1-CRE (5'-TAACGTCA-3') for binding transcription factors and reduced cyclin D1 gene expression (in reporter gene assay, Northern blotting and Western blotting) to modulate cyclin D1/Cdk4/pRB signaling and G1-S progression in a steady state and/or under estrogen stimulation. Decrease of cyclin D1 protein level by CRE-decoy treatment was also observed in p53-mutated cancer cells. Cyclin D1 expression was also diminished in MCF7 cells stably expressing dominant negative mutant CREB indicating that the nonspecific effect of oligonucleotide or its degradation products could be excluded. These data suggest that inhibition of cyclin D1 expression contributes to the growth inhibition induced by the decoy oligonucleotide in MCF7 cells through a cyclin D1/Cdk4/pRB signaling pathway. Downregulation of cyclin D1 expression also provides a mechanism of CRE-decoy-induced growth inhibition in tumor cells having p53 mutation.

Breast Neoplasms↗

PubMed2002

Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.

Amino Acid Substitution↗

PubMed1992

Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction.

The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.

Chromosome Inversion↗

PubMedDate not supplied

Trypanosoma brucei: a first-generation CRE-loxP site-specific recombination system.

The bacteriophage CRE-loxP system of DNA recombination is widely used to manipulate segments of the genomes of mice and other eukaryotes for the purpose of studying the regulation and functions of their genes. Since this recombination system could have similar applications in analyzing the genomes of trypanosomatids, we assessed the action of CRE recombinase on its loxP DNA recognition sites in Trypanosoma brucei after inserting tetracycline-regulated CRE and two 34-bp loxP sites into the T. brucei genome. We found that when loxP sites flank in a direct orientation the transcription termination sequence (1.1 kb) of the T. brucei GPEET/PAG3 locus, CRE recombinase deletes this termination sequence, permitting transcription and subsequent expression of a downstream reporter gene for the green fluorescent protein (GFP). Thus, the CRE-loxP system is highly efficient in T. brucei, but the experimental results also indicate that a better way than the existing tetracycline-regulated system is required to completely silence expression of CRE in the T. brucei genome when it is not needed before the full range of CRE-loxP applications currently used in mice can be exploited in African trypanosomes.

Animals↗

PubMed2000

Developmental profile of kainate receptor subunit KA1 revealed by Cre expression in YAC transgenic mice.

To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.

Aging↗

PubMed1990

DNA specificity of the Cre recombinase resides in the 25 kDa carboxyl domain of the protein.

The Cre protein of bacteriophage P1 is a 38.5 kDa site-specific recombinase that belongs to the Int family of recombination proteins. Cre acts by binding specifically to a 34 base-pair sequence, lox, where it carries out recombination. A limited chymotryptic digest of Cre resulted in two fragments of sizes 25 and 13.5 kDa, respectively. The sequence of the amino terminus of the purified 25 kDa peptide demonstrates that this peptide represents the carboxyl-terminal portion of the Cre protein. A truncated version of the cre gene was constructed which produces only the 25 kDa peptide. The 25 kDa peptide is capable of specific binding to the lox site, but binds at lower affinity than does wild-type Cre. Footprinting with Fe-EDTA indicates that the 25 kDa peptide protects the inverted repeats of the lox site but shows only partial protection of the spacer region. This is in contrast to the footprint obtained with wild-type Cre which protects the entire spacer region.

Amino Acid Sequence↗

PubMed2002

Cre-loxP biochemistry.

Cre recombinase is now widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. For in vitro experiments, there is a clear need for highly pure preparations of Cre and of Cre mutants that serve as controls or supply an altered activity or specificity. In vivo experiments utilizing Cre variants also often require in vitro characterization and some applications involve transfection of purified enzyme to achieve transient activity in the cell. This review outlines a detailed protocol for purification of native Cre and describes straightforward assays that can be used to test for recombination activity in vitro. The design of experiments to trap the intermediates of Cre-loxP site-specific recombination for biophysical studies is also presented. The methods described should be useful to any investigator with a need for purified Cre recombinase and should be broadly applicable to related site-specific recombination systems.

DNA↗

PubMed2003

Modulation of the active complex assembly and turnover rate by protein-DNA interactions in Cre-LoxP recombination.

Cre promotes recombination at the 34 bp LoxP sequence. Substitution of a critical C-G base pair in LoxP with an A-T base pair, to give LoxAT, reduced Cre binding in vitro and abolished recombination in vivo [Hartung, M., and Kisters-Woike, B. (1998) J. Biol. Chem. 273, 22884-22891].We demonstrated that LoxAT can be recombined in vitro. However, Cre discriminates against this substrate both before and after DNA binding. The preference for LoxP over LoxAT is the result of reduced binding and a slower turnover rate, amplified by changes in cooperativity of complex assembly. With LoxAT, similar levels of substrate turnover required 2-2.5-fold higher protein-DNA concentrations compared to LoxP, but the sigmoidal behavior of the concentration dependence was more pronounced. Further, the Cre-LoxAT complexes reacted 4-5-fold more slowly. In the 2.3 A resolution Cre-LoxAT complex structure, the major groove Arg259-guanine interaction was disrupted, explaining the reduced binding. Overall structural shifts and mobility changes indicate more favorable interactions between subunits, providing a hypothesis for the reduced turnover rate. Concomitant with the displacement of Arg259 from the DNA, adjacent charged residues Glu262 and Glu266 shifted to form salt bridges with the Arg259 guanidinium moiety. Substitution of Glu262 and Glu266 with glutamine increased Cre complex assembly efficiency and reaction rates with both LoxAT and LoxP, but diminished Cre's ability to distinguish them. The increased rate of this variant suggests that DNA substrate binding and turnover are coupled. The improved efficiency, made at some expense of sequence discrimination, may be useful for enhancing recombination in vivo.

Amino Acid Substitution↗

PubMed1998

Efficient BLG-Cre mediated gene deletion in the mammary gland.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

Animals↗

PubMed1998

Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning.

Recent studies suggest that the CREB-CRE transcriptional pathway is pivotal in the formation of some types of long-term memory. However, it has not been demonstrated that stimuli that induce learning and memory activate CRE-mediated gene expression. To address this issue, we used a mouse strain transgenic for a CRE-lac Z reporter to examine the effects of hippocampus-dependent learning on CRE-mediated gene expression in the brain. Training for contextual conditioning or passive avoidance led to significant increases in CRE-dependent gene expression in areas CA1 and CA3 of the hippocampus. Auditory cue fear-conditioning, which is amygdala dependent, was associated with increased CRE-mediated gene expression in the amygdala, but not the hippocampus. These data demonstrate that learning in response to behavioral conditioning activates the CRE transcriptional pathway in specific areas of brain.

Animals↗

PubMed2001

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.

The use of Cre/loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER(T) show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.

Aneuploidy↗