Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination

We have examined the linking-number changes that occur during phage P1 Cre-mediated recombination in vitro between two loxP sites. Such recombination reactions can be divided into three types: intramolecular inversion, in which recombination occurs between two loxP sites in opposite orientations on the same DNA substrate; intramolecular excision, where recombination occurs between two loxP sites that are in the same orientation on the DNA substrate; and intermolecular recombination, which occurs between two loxP sites on separate DNA molecules. Our results indicate that inversion changes the linking number of the substrate DNA by two topological turns. With a negatively supercoiled substrate, the product is changed by +2 turns. A relaxed substrate yields products that have been changed by either +2 or -2 turns. For intermolecular reactions, the sum of the linking numbers of each of the two starting circles is equal to the linking number of the dimer circle generated by recombination, and no change occurs in linking number. For intramolecular excision reactions, the data are most consistent, with no change in linking number during recombination. These results are discussed in terms of models for alignment and synapsis of the recombining sites and the mechanism of strand exchange.     

Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.     

Cre-mediated recombination in mouse Clara cells

Clara cells are nonciliated secretory cells lining the respiratory epithelium and are easily identified by the expression of Clara cell secretory protein (CCSP). To investigate molecular mechanism(s) regulating Clara cell function in the lungs, Cre recombinase was inserted into exon 1 of the CCSP, generating two novel mouse models, CCSP(Cre-Neo) and CCSP(Cre). These two models differ only by the inclusion of the neomycin resistance gene. These mice were bred to the R26R reporter mouse to investigate the tissue and cell specificity of Cre-mediated recombination. The efficiency of Cre recombination in the CCSP(Cre) mouse model was higher than in the CCSP(Cre-Neo) mouse model. Recombination was detected at D 4.5 in CCSP(Cre-Neo)/R26R mice and at D 0.5 in CCSP(Cre)/R26R mice. The CCSP(Cre-Neo) and CCSP(Cre) mouse models provide valuable tools for the ablation of genes in the postnatal mouse Clara cells.     

Cre-mediated recombination in the pituitary gland

Organ-specific expression of a cre recombinase transgene allows for the analysis of gene function in a particular tissue or cell type. Using a 4.6 kb promoter from the mouse glycoprotein hormone alpha-subunit (alphaGSU or Cga) gene, we have generated and characterized a line of transgenic mice that express cre recombinase in the anterior and intermediate lobes of the pituitary gland. Utilizing a cre-responsive reporter transgene, alphaGSU-cre transgene expression was detected in the pituitary primordium and in all five cell types of the adult anterior pituitary. alphaGSU-cre transgene activity was also detected in the cardiac and skeletal muscle. Little or no activity was evident in the gonads, adrenal glands, brain, ventromedial hypothalamus, or kidneys. The alphaGSU-cre transgenic mice characterized here will be a valuable tool for examining gene function in the pituitary gland.     

Specificity and efficiency of Cre-mediated recombination in Emx1-Cre knock-in mice

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.     

Cre-mediated recombination in rhombic lip derivatives

To study the development of the cerebellum, we generated a transgenic mouse line Tg(malpha6-cre)B1LFR that expresses CRE recombinase under the GABA(A) receptor alpha6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be explained by a very early specification event at the rhombic lip that primes cells derived from this structure to express the transgene during neuronal maturation. As no recombination occurred in the inferior olive, it may be derived from a distinct subset of precursors at the rhombic lip. No recombination occurred in any of the interneurons in the cerebellum (stellate cells, basket cells, and Golgi cells), consistent with the hypothesis that they are not derived from the same embryonic tissue as the granule cells.     

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T₀-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Cre-mediated recombination in the skin melanocyte lineage

Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.     

Site-specific transgenesis by Cre-mediated recombination in Drosophila

Transposons such as P elements are routinely used to stably transfer exogenous DNA (transgenes) into the Drosophila genome. Transgene insertion events, however, are essentially random and are subject to 'position effects' from nearby endogenous regulatory elements. Here we describe a microinjection-based system that uses Cre-mediated recombination to insert transgenes into precise genomic 'landing sites'. The system is simple and efficient, and will permit precise comparisons between multiple transgenic constructs.     

Cre-mediated site-specific gene integration for consistent transgene expression in rice

To minimize expression variability amongst transgenic lines, we have utilized the strategy of Cre/lox-mediated site-specific gene integration. This method allows the precise integration of a transgene in a lox site previously placed in the genome. Using the biolistic method for DNA delivery, we have generated several site-specific integrant lines, derived from three different target lines. About 80% of the selected lines contain precise integration of the gusA reporter gene and fall into two categories: single-copy (SC) lines that contain site-specific integration without additional random integrations, and multicopy (MC) lines that contain random integrations in addition to the site-specific integration. The expression of the gusA gene was studied in callus cells and regenerated plants. The isogenic SC lines displayed significantly lower expression variation, whereas much higher expression variation was observed in MC lines. Furthermore, stable inheritance of the gusA gene was observed in T1 plants derived from a subset of SC lines. This demonstrates that consistent gene expression can be obtained in rice by Cre-mediated site-specific integration.     

Measurements of DNA-loop formation via Cre-mediated recombination

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.     

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

Background  

Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.     

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.     

Tamoxifen dosing for Cre-mediated recombination in experimental bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time–points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2 mg applied to newborn mouse pups in 10 μl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease.     

Dual renin gene targeting by Cre-mediated interchromosomal recombination

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.