Carbonic anhydrase i car1 is a gene expressed by colonic epithelial cells. Cre lox recombination is a sitespecific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the dna of cells. Expression patterns of inducible cre recombinase driven by. Hypoxiainducible factor1 alpha maintains mouse articular. These transgenic mice express a tamoxifen inducible cre recombinase driven by the mouse col1a2, collagen, type i, alpha 2, promoter.
The cre lox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. Inducible cre mice are very useful to study the function of genes in specific tissues. Frontiers promoter specificity and efficacy in conditional. Temporal genetic modification of mice using the ligand inducible cre loxp system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes.
Modification of gene activity in mouse embryos in utero by a. Transgenic mice expressing a ligand inducible cre recombinase in osteoblasts and odontoblasts. Inducible gene inactivation in neurons of the adult mouse. Hypoxia inducible factor1 alpha maintains mouse articular cartilage through suppression of nf. Inducible gene targeting in the neonatal vasculature and. Creert has a preference for tamoxifen over estrogen but the. In order to achieve temporally controlled somatic mutagenesis, the cre loxprecombination system has been complemented with the tamoxifen inducible fusion protein.
A creinducible diphtheria toxin receptor mediates cell. Pdf tamoxifen tminducible cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Conditional and inducible transgene expression in endothelial and. The two major induction systems are tetracycline and tamoxifen.
Pdf functional study of gene using inducible cre system. The advantage of combining the tetracyclineinducible and creloxp systems is. In this study we focused on tamoxifen degradation kinetics, because for all genetic fatemapping studies, the period during which tamoxifen or its metabolites remain active. Inducible fli1 gene deletion in adult mice modifies several myeloid lineage commitment decisions and accelerates proliferation arrest and terminal erythrocytic.
Modification of gene activity in mouse embryos in utero by a tamoxifen inducible form of cre recombinase. The analysis of the role of genes in important brain functions like learning, memory and synaptic plasticity requires gene inactivation at the adult stage to exclude developmental effects, adaptive changes or even lethality. The data demonstrate essentially no cremediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but 80% recombination after only four tamoxifen injections. Tamoxifen administration allows for ablation of predetermined genes in. A transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult. Compared with the constitutively active cre, inducible creer allows external. Nephrin is necessary for podocyte recovery following injury in an adult.
Generation of inducible cre systems for conditional gene. Conditional and inducible transgene expression in endothelial. Conditional knockout mice are then generated by breeding the. Time of initiation of expression of the cre recombinase eightweekold adult mice for dat. Inducible cre transgenic mouse strain for skeletal muscle.
Transgenic construct nsemercremer was constructed by linking the rat nse promoter 5. The modification of the mouse genome by sitespecific gene insertion of transgenes and other genetic elements allows the study of gene function in different developmental stages and in the pathogenesis of diseases. Neoplastic transformation was induced in 8 to 12weekold mice by tailvein injection of 10 9 infectious units of adenovirus expressing cre recombinase vector biolabs. The stop cassette, which prohibits dtr expression, is removed by crossing the idtr strain to a tissuespecific cre expressing mouse strain. Pcr analysis and by crossbreeding to zeg reporter mice. Tamoxifen inducible cre mediated manipulation of animal genomes has achieved wide acceptance over the last decade, with numerous important studies heavily relying on this technique. Nephrin is necessary for podocyte recovery following injury in an adult mature glomerulus. Pdf cardiacspecific inducible and conditional gene.
An inducible hepatocellular carcinoma model for preclinical. A new tool to examine physiology and disease of postnatal bone and tooth. Generation of nsemercremer transgenic mice with tamoxifen. Inducible transgenic mouse models allow for the activation of genes in specific cells and tissues at specific times. The transgene insert contains a fusion product involving cre recombinase and a mutant form of the mouse estrogen receptor ligand binding domain. Podocytespecific expression of tamoxifen inducible cre recombinase in mice podocytespecific expression of tamoxifen inducible cre recombinase in mice. Treatment with tamoxifen leads to a fibroblastspecific deletion of exon 4 of the tbrii protein. May 20, 2005 a general scheme of the inducible dtr mouse strain idtr. When crossed with a strain containing a loxp site flanked sequence of interest, the offspring are useful for generating tamoxifeninduced, cre mediated targeted deletions.
Highperformance chemical and lightinducible recombinases in. Pdf the crelox sitespecific recombination system has emerged as an important tool for the. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis. Many transgenic mouse lines have been generated to obtain astrocytespecific expression of inducible cre recombinase for functional studies.
Inducible gene targeting based on the cre loxp system is a novel technique allowing conditional gene modification in mice. Generation of inducible cre systems for conditional. Podocytespecific expression of tamoxifeninducible cre. The purpose of this study was to create and characterize an inducible, skeletal musclespecific cre transgenic mouse strain. Temporally regulated and tissuespecific gene manipulations. To quantify the gene deletion efficiency that can be achieved in lung epithelial cells using lung epithelial cellspecific, doxinducible cre deleter mice, both the activator spcrtta and ccsprtta and operator tetocre stg lines were crossed to rosa26 yfpyfp reporter stg mice to generate dtg mice as shown in mating i, figure 1a. Podocytespecific tetracycline inducible cre transgenic mice are constructed using nphs2 promoter. Using our orthogonal inducible recombinases, we created a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell. In the present study we observed a previously unreported focal fibrosis and depressed leftventricular. They provide trophic support for neurons, modulate synaptic transmission and plasticity, and contribute to neuronal dysfunction. Schutz german cancer research center, heidelberg, germany, which contains nuclear localization signal cre, the mutated lbd of human progesterone receptor amino acids 641 to 891, 12 and the simian virus sv 40 polyadenylation signal.
Expression of cre in presence or absence of ligand. Tamoxifeninducible gliaspecific cre mice for somatic. Rapid and highly efficient inducible cardiac gene knockout in. Inducible cre transgenic mouse strain for skeletal. However, many complete and tissuespecific cremediated knockout mice result in embryonic or early perinatal death, thereby precluding the analysis of gene. Tissuespecific and inducible cre mediated recombination in the gut epithelium. Tamoxifenindependent recombination of reporter genes limits. Generation of an inducible colonspecific cre enzyme mouse.
Oct 24, 2019 the availability of high performance recombinases with low basal activity and high dynamic range is limited. Pdf the use of the creloxp system for gene targeting has been proven to be a powerful tool for. Tamoxifen tm inducible cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. The col1a2creer t mice were crossed with the tbrii floxed mice. Dec 11, 2019 astrocytes are the most abundant cell type in the central nervous system cns.
Transgenic mice, inducible gene knockout, somatic mutagenesis, creer recombinase. Aug 29, 2019 furthermore, we have engineered inducible split cre systems with better performance than existing inducible cre systems. However, the commonly used myh6mercremer system requires timeconsuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. Transgenic mice expressing a ligandinducible cre recombinase in osteoblasts and odontoblasts. When these tgcsf1rmericremer1jwp mice are bred with mice containing a loxpflanked sequence of interest, tamoxifen inducible, cre mediated recombination will result in deletion of the flanked sequences in csf1rexpressing cells. Here the authors present a library of over 20 orthogonal split recombinases that can be. Oct 20, 2016 the tamoxifen inducible cre loxp system is widely used to overcome gene targeting preadult lethality, to modify a specific cell population at desired timepoints, and to visualize and trace cells in fatemapping studies.
Efficiency and specificity of gene deletion in lung. Tamoxifen inducible cre system is achieved by modified cre protein fused. Pdf inducible fli1 gene deletion in adult mice modifies. In this study, we generated a tamoxifen inducible cre driver mouse strain for the purpose of widespread and temporal cre recombination.
These transgenic mice have a tamoxifen inducible cre mediated recombination system driven by the mouse plp1, proteolipid protein myelin 1 promoter. This requires a transgenic mouse in which the gene of. It allows the dna modification to be targeted to a specific cell type or be triggered by a specific external stimulus. Tissuespecific and inducible cremediated recombination. Efficient recombination in diverse tissues by a tamoxifen. Using a plpcreert2 transgene, we have generated mice that show specific inducible cre function, as analyzed by crossbreeding experiments into the.
We generated an inducible car1 creer mouse model with cre expression in the cecum and. Uncovering the fine print of the creert2loxp system while. The use of the creloxp system for gene targeting has been proven to be a powerful tool for understanding gene function. The creestrogen receptor er is expressed under the control of the fibroblastspecific col1a2 promoter. Pdf tamoxifeninduced creloxp recombination is prolonged. Establishment of a tamoxifeninducible credriver mouse. Precondition of this method is the regulation of cre activity by inducible genetic switches. Cre inducible ast transgenic mice bred in the c57bl6 background were housed in the institutional animal facility. Cardiac fibrosis in mice expressing an inducible myocardial.
Inducible transgenesis provides a valuable technique for the analysis of gene function in vivo. When these caggcreer tm mice are bred with mice carrying a floxed target gene, tamoxifen inducible cre mediated recombination results in deletion of the floxed sequences in widespread cells or tissues of the offspring, including developing embryos and cultured cells derived from transgenic mice. Modification of gene activity in mouse embryos in utero by. The crelox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. Here, we generated a genomic safe harbor hipp11 h11 locusspecific knockin transgenic mouse line in which the albumin promoter is used to drive the expression of the. Expression and activity of cre was evaluated using reverse transcriptase polymerase chain reaction rt. Conditional and inducible transgene expression in mice through the combinatorial use of cre mediated recombination and tetracycline induction. Oct 18, 2016 a major limitation of current mouse models of colorectal cancer crc is that the cancer that develops is often significantly different from human colon cancer in terms of latency, intestinal location, or molecular signature. Although creloxp systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of macrophage targeting cre lines. Highperformance chemical and lightinducible recombinases. Recently, a number of groups have reported transient complications of using this protocol in the heart. Cardiacspecific inducible and conditional gene targeting in mice. In this paper, we provide a detailed protocol for tamoxifen inducible gene deletion in neonatal mice, as well as for retina dissection, wholemount immunostaining and the quantitation of ec. Tamoxifeninducible gliaspecific cre mice for somatic mutagenesis.
Tamoxifeninduced creloxp recombination is prolonged in. These transgenic mice were crossed with the rosa26 laczfloxtargeted mice to examine cre recombinase activity and the fidelity of the system. Inducible gene targeting in mice using the cre loxp system has become a valuable tool to analyse the roles of specific genes in the adult heart. Comparing our results from tamoxifen inducible datcreert2 transgenic mice with the dat cre knockin mice, it was clear that stronger tdtom reporter expression was observed on recombination achieved by the tamoxifen inducible datcreert2 than the dat cre. Inducible gene targeting in postnatal myocardium by cardiac. Nov 24, 2017 conditional and inducible creloxp systems are used to target gene deletion to specific cell lineages and tissues through promoterrestricted expression of the bacterial dna recombinase, cre. We report the generation and characterization of mouse lines carrying glia lineagespecific transgenes expressing an improved variant of the tamoxifen inducible cre recombinase, creert2, where the recombinase is fused to a mutated ligand binding domain of the human estrogen receptor. Additionally, creloxp allows the inducible overexpression of transgenes or reporters. To achieve skeletal musclespecific expression, the human. Although the efficiency of inducible cre loxp recombination can be easily evaluated with reporter strains, the precise length of time that tm induces nuclear translocation of creertm and subsequent recombination of a target allele is not well defined, and difficult to. Use of temporalregulated cre transgenic mice is one option in investigating the role of the gene in the postnatal or diseased organs.
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