Cre Recombinase Protein Research Articles

Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.

Selective expression of an aP2/Fatty Acid Binding Protein 4-Cre transgene in non-adipogenic tissues during embryonic development.

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase

The biomedical application of human embryonic stem (hES) cells will increasingly depend on the availability of technologies for highly controlled genetic modification. In mouse genetics, conditional mutagenesis using site-specific recombinases has become an invaluable tool for gain- and loss-of-function studies. Here we report highly efficient Cre-mediated recombination of a chromosomally integrated loxP-modified allele in hES cells and hES cell-derived neural precursors by protein transduction. Recombinant modified Cre recombinase protein translocates into the cytoplasm and nucleus of hES cells and subsequently induces recombination in virtually 100% of the cells. Cre-transduced hES cells maintain the expression of pluripotency markers as well as the capability of differentiating into derivatives of all three germ layers in vitro and in vivo. We expect this technology to provide an important technical basis for analyzing complex genetic networks underlying human development as well as generating highly purified, transplantable hES cell-derived cells for regenerative medicine.

A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination

BackgroundCre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants.ResultsWe created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase.ConclusionThis study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.

Genetic Evidence That an 11 Amino Acid β-Hairpin Loop in the Cytoplasmic Domain of Band 3 Is Responsible for at Least 50% of Ankyrin Binding in Mouse Erythrocytes.

The anion exchange protein (band 3) is an integral erythrocyte membrane protein that is associated with the spectrin-actin cytoskeleton through ankyrin, which binds to both band 3 and spectrin. Improper band 3/ankyrin interactions result in erythrocyte instability leading to spherocytosis and hemolytic anemia. The crystal structures of ankyrin and band 3 predict that the ankyrin amino-terminus, containing comb-like ANKYRIN repeat subdomains, binds to a β-hairpin loop (residues 175-185) in the band 3 cytoplasmic domain. We have previously shown that replacement of human band 3 residues 175–185 with a di-glycine bridge eliminates the binding of an ankyrin fragment containing domains 3 and 4 to the cytoplasmic domain of band 3 in vitro. To test this model for ankyrin/band 3 interactions in vivo, we used homologous recombination in embryonic stem cells to replace the corresponding mouse band 3 sequences, residues 189–199 in exon 7, with a di-glycine bridge to create a knock-in transgenic mouse. Analysis of mRNA expression in transgenic mice carrying the targeted locus demonstrated that the level of mRNA from the mutant allele (which contains the neomycin resistance gene used for positive selection of embryonic stem cells) was 50-70 fold lower than the level of mRNA from the wild type allele. Heterozygous transgenic mice were mated to mice expressing the Cre recombinase protein in the germ line to remove the neomycin resistance gene. After removal of the neomycin resistance gene from the band 3 locus, mice homozygous for the 189–199 deletion/di-glycine insertion were born in a normal Mendelian ratio and had normal numbers of red blood cells, white blood cells and platelets. Quantitative RT-PCR analysis showed that the level of band 3 mRNA from the wild type and mutant alleles were identical. SDS poly acrylamide gel electrophoresis and Western Blot analysis demonstrated normal levels of band 3 and other red cell membrane proteins in wild type, heterozygous and homozygous mutant erythrocytes. Homozygous mutant mice showed a modest but significant increase in osmotic fragility (p<0.01) compared to wild type or heterozygous mice. Ankyrin binding to inside out vesicles (IOV) from wild type, heterozygous and homozygous mutant erythrocytes was measured using an 125I labeled peptide containing domains 3 and 4 of mouse ankyrin. Ankyrin binding to homozygous mutant IOVs was 50% or less than the level of ankyrin binding to WT IOVs. We conclude from these studies that the 11 amino acid β-hairpin loop encoded by residues 189–199 of the band 3 gene is responsible for at least 50% of the ankyrin binding in mouse erythrocytes. We predict that mutations in this region of human band 3 would lead to a phenotype of Hereditary Spherocytosis similar to that seen with ankyrin deficiency. Space filling models of the cytoplasmic domain of band 3 have identified a second homologous β-hairpin loop encoded by residues 63-75, which is the target of our future investigation.

Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud

Fibroblast growth receptors (FGFRs) consist of four signaling family members. Mice with deletions of fgfr1 or fgfr2 are embryonic lethal prior to the onset of kidney development. To determine roles of FGFR1 and FGFR2 in the ureteric bud, we used a conditional targeting approach. First, we generated transgenic mice using the Hoxb7 promoter to drive cre recombinase and green fluorescent protein expression throughout ureteric bud tissue. We crossed Hoxb7creEGFP mice with mice carrying lox-p sites flanking critical regions of fgfr1 and/or fgfr2. Absence of fgfr1 from the ureteric bud ( fgfr1 UB−/− ) results in no apparent renal abnormalities. In contrast, fgfr2 UB−/− mice have very aberrant ureteric bud branching, thin ureteric bud stalks, and fewer ureteric bud tips. Fgfr2 UB−/− ureteric bud tips also demonstrate inappropriate regions of apoptosis and reduced proliferation. The nephrogenic mesenchymal lineage in fgfr2 UB−/− mice develops normal-appearing glomeruli and tubules, and only slightly fewer nephrons than controls. In contrast, fgfr2 UB−/− kidneys have abnormally thickened subcapsular cortical stromal mesenchyme. Ultimately, fgfr2 UB−/− adult kidneys are small and abnormally shaped or are hydronephrotic. Finally, there are no additional abnormalities in the fgfr1/2 UB−/− kidneys versus the fgfr2 UB−/− kidneys. In conclusion, FGFR2, but not FGFR1, appears crucial for ureteric bud branching morphogenesis and stromal mesenchyme patterning.

Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination.

Development of efficient short-term gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study, we present a highly efficient, nonviral non-DNA technology for genetic loading of mouse ES cells based on electroporation of defined mRNA. Here, we show that mouse ES cells can be efficiently loaded with mRNA encoding a green fluorescent reporter protein, resulting in a level of at least 90% of transgene expression without loss of cell viability and phenotype. To show that transgenes, introduced by mRNA electroporation, exert a specific cellular function in transfected cells, we electroporated stably transfected ES cell lines with mRNA encoding FLPe or Cre recombinase proteins in order to excise an FRT- or LoxP-flanked reporter gene. The results, as determined by the disappearance and/or appearance of a fluorescent reporter gene expression, show that FLPe and Cre recombinase proteins, introduced by mRNA electroporation, efficiently exert their function without influence on further culture of undifferentiated ES cell populations and their ability to differentiate towards a specific lineage.

Site-specific recombination induced in transgenic plants by PVX virus vector expressing bacteriophage P1 recombinase

Potato Virus X (PVX)-based vector has been constructed for transient expression of Cre recombinase protein in lox-containing transgenic Nicotiana benthamiana plants. In the virus genome, the cre gene was placed under the transcriptional control of the viral coat protein subgenomic promoter. The resulting recombinant virus (PVX-Cre) expressed functional Cre which mediated the excision of a lox-flanked sequence in the plant genome. PVX-Cre-infected plants were self-pollinated and the progeny was tested for the inheritance of site-specific recombination. One of the three plants tested transmitted the recombined state to all progeny. These results show for the first time that a recombinant plant virus vector produced a functional Cre recombinase. PVX-Cre-mediated recombined loci can be germinally transmitted.

Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins.

Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins.

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.

Induced DNA recombination by Cre recombinase protein transduction.

Cre is a DNA recombinase that recognizes 34 base-pair loxP sites of recombination. We have developed a cell-permeable Cre recombinase, TATCre, that is capable of mediating deletion of loxP-flanked targets by simply adding TATCre to cell cultures. Thus, TATCre allows efficient induced DNA recombination without the use of a Cre recombinase transgene or any other genetic material and should prove useful for the genetic manipulation of a wide variety of cell types that have been engineered to possess loxP sites.

Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.

The advent of conditional and tissue-specific recombination systems in gene-targeted or transgenic mice has permitted an assessment of single gene function in a temporally regulated and cell-specific manner. Here we generated transgenic mice expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the alpha-myosin heavy chain promoter. These transgenic mice were crossed with the ROSA26 lacZ-flox-targeted mice to examine Cre recombinase activity and the fidelity of the system. The data demonstrate essentially no Cre-mediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but >80% recombination after only four tamoxifen injections. Expression of the MerCreMer fusion protein within the adult heart did not affect cardiac performance, cellular architecture, or expression of hypertrophic marker genes, demonstrating that the transgene-encoded protein is relatively innocuous. In summary, MerCreMer transgenic mice represent a tool for temporally regulated inactivation of any loxP-targeted gene within the developing and adult heart or for specifically directing recombination and expression of a loxP-inactivated cardiac transgene in the heart.

Visions of IFT

We see the world through cilia. The outer segments of rods and cones are highly specialized sensory cilia, and the entire photosensing machinery including opsin is located in the outer segment and must be transported through a narrow connecting cilium that separates the outer segment from the cell body. But what motors drive this transport? A kinesin-driven motility called intraflagellar transport (IFT) was first discovered in the motile flagella of the unicellular green alga Chlamydomonas. IFT was found to be driven by a heterotrimeric kinesin-II motor. IFT has also been observed in the olfactory cilia of the nematode Caenorhabditis elegans, and again a heterotrimeric kinesin-II is required to drive this motility. Given that flagella and cilia from such two diverse species rely on the same kinesin-II-driven IFT process for transport, is it possible that IFT might also be needed in the connecting cilium of retinal photoreceptor cells?To investigate the role of IFT specifically in photoreceptor cells, Marszalek and colleagues1xGenetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Marszalek, J.R. et al. Cell. 2000; 102: 175–187Abstract | Full Text | Full Text PDF | PubMedSee all References1 constructed mice in which the gene encoding KIF3A, one of the kinesin-II subunits, contained a pair of loxP site-specific recombination sequences that, when excised, caused a frameshift owing to removal of one exon, thus leading to a completely nonfunctional protein sharing only the first methionine with the original protein. The Cre recombinase protein was then specifically expressed in rod cells under control of a photoreceptor-specific promoter.Loss of KIF3A caused extensive cell death among photoreceptor cells, leading to loss of as many as 80% of all rod cells. Ultrastructural analysis revealed that protein and vesicles accumulated at the base of the connecting cilium, suggesting that there might be a defect in transport of this material into the outer segment. Immunogold electron microscopy revealed that opsin is found in these deposits, showing that loss of KIF3A leads to a defect in opsin transport through the connecting cilium.Because defects in opsin transport are implicated in retinal degeneration diseases, might the cell death be caused by the defect in opsin transport? One hint that this could be true came from the fact that opsin mislocalization was observed prior to any observable cell death. To confirm this, retinas from the KIF3A conditional knockout mice were stained by immunofluorescence to detect opsin accumulation and simultaneously stained using a TUNEL assay to visualize apoptosis. It was found that 90% of all apoptotic cells showed defects in opsin localization, but a large fraction of cells with opsin accumulation defects did not show any signs of apoptosis. This implies that opsin accumulation must precede apoptosis.These results show that IFT is crucial for photoreceptor cell maintenance and suggest that defects in this transport process might underlie a range of retinal degeneration diseases in humans.

V(D)J recombination is not activated by demethylation of the kappa locus.

V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.

CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR ENCODING Fas LIGAND WITH AN INDUCIBLE SYSTEM

166 Immune privilege sites like the eye, testis and brain have been recognized that express Fas Ligand (FasL) allowing foreign agents and tissues to persist in those locations, this phenomenon has long held the promise of solving the problems of autoimmunity, and graft rejection. New studies challenges the immunoprotective effect of FasL, adenovirus-mediated expression of FasL in pancreatic islet allografts induced neutrophilic infiltration and islet destruction suggesting that probably other molecules may also be involved. In addition, the magnitude and temporal pattern of FasL expression may be key determinants of its efficacy in this context Finally, naturally occurring alleles of FasL have different abilities to trigger apoptosis through Fas, suggesting that polymorphism of FasL affects the biological activity. Expression of FasL in cells with intrinsic expression of Fas (hepatocytes, islets, adenoviral packaging cell line 293, etc) results in an autocrine loop that induces apoptosis. To further characterize the role of Fas L in rejection and autoimmunity, we decided to develop an adenovirus vector expressing FasL in an inducible system. Methods: Our recombinant adenovirus is based upon the Cre/Loxp system. In this vector the mFasL is functionally separated from the modified chicken β-actin promoter with the cytomegalovirus immediate-early enhancer (CAG) by a Loxp-flanked stuffer segment. The encoded Cre recombinase protein would be predicted to excise the stuffer and allow functional of the expression cassette at the deleted E1 site of the recombinant adenovirus vector. Thus, this strategy based on inducible gene expression offered the means to construct such a vector expessing FasL, regardless of the expression of Fas by the viral vector packaging cell line. The recombinant adenovirus AdLoxpFasL was constructed by the two-plasmid homologous recombination method of Parks et al. As a control in all experiment an adenovirus expressing an irrelevant virus was used.RESULTS: Plaques were picked approximately 10 days posttransfection and carried through three additional isolation steps. Adenovirus genome DNA was subjected to restriction endonuclease digestion to confirm their identity. Southern and Northern Blot was preformed with a32 P-labeled mFasL probe. No leaking of the system was detected. The functionality of this construct was evaluated by 51Cr-release assay in which a FasL sensitive cell line (A20) was used as a target cell, and a increase cell killing was observed when increased the E/T ratio. Adult female C57BL/6 and B6 lpr/lpr mice were injected via the tail vein with adenovirus vector constructs. Twenty-four hours after injection only the C57BL/6 mice that received the AdLoxpFasL plus the AxCANCre vectors demonstrated significant inflammatory influx and apoptosis in hepatic parenchyma. These changes were not seen in the groups that received AdCMVLacZ and AdLoxpFasL or in the control group that received no virus. CONCLUSIONS: In this study we demonstrated that the AdLoxpFasL vector is capable of expressing high levels of mFasL in target cells under the control of Cre recombinase This Adenovirus vector encoding mFasL in an inducible system will allow further evaluation of the role of FasL in autoimmunity and allograft rejection.

CRE-mediated recombination at the murine whey acidic protein (mWAP) locus

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. The types of recombination products generated in a wild-type Cre reaction can also be affected by mutations in the loxP site itself. Altered sites have been identified that are recombined by wild-type Cre to produce many topologically complex products. These altered sites cannot be recombined by Cre111. These observations are consistent with the idea that the Cre111 mutant is altered in synapsis. Whether this accounts for the slow rate of recombination is not clear at present.

Genomic targeting with an MBP-Cre fusion protein

The Cre recombinase of bacteriophage P1 catalyses site-specific recombination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic research. Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombinase protein rather than a cre-expression plasmid is used to direct site-specific recombination (Baubonis and Sauer (1993) Nucleic Acids Res., 21, 2025–2029). We bacterially produced a Cre recombinase containing a nuclear localisation signal as a fusion protein with the E. coli maltose binding protein (MBP) and purified the protein by one step affinity chromatography. Subsequent cleavage with the protease factor Xa releases the Cre recombinase including the nuclear localisation signal from the maltose binding protein. Surprisingly, we found that the recombination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase. This suggests that the MBP portion of the fusion protein behaves as a separate protein domain which does not interfere with Cre activity and can thus be used as an independent molecular tag. Additionally, the fusion protein is very resistant to proteolytic degradation and active over a wide range of temperatures. It efficiently catalyses excision and integration reactions in vitro and in eukaryotic cells. Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DNA sequence from a plasmid and integrate it into a pre-existing lox site in the genome in one transfection experiment. Vector backbone sequences which might have undesirable effects can thereby be excluded. The MBP-Cre fusion protein described here will be a useful tool not only for the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific recombination.

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.

Genomic targeting with purified Cre recombinase.

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.

Site-directed recombination in the genome of transgenic tobacco

The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.