The method uses an interferonresponsive promoter to control the expression of cre recombinase. System for tamoxifeninducible expression of crerecombinase. Abstract cre and creer mouse strains are powerful tools that have. 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. Moon1,4,5 to study the roles of key transcription factor networks, growth factors, and signaling molecules in the endoderm, notochord, and.
Cardiac fibrosis in mice expressing an inducible myocardial. The number of breeding steps required to derive experimental animals and the expected yield in percent of offspring according to mendelian inheritance is also given. 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 creer tm cdna was placed under the regulation of the wnt1 enhancer. 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. Inducible gene targeting in mice using the cre lox system brian sauer laboratory of biochemistry and metabolism, national institute of diabetes, digestive and kidney disease, national institutes of health, building 10, room 9n119, bethesda, maryland 208921800 email. The cre lox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. Breeding scheme for the generation of reporter mice for the detection of cre activity. 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. The use of the cre loxp system for gene targeting has been proven to be a powerful tool for understanding gene. Thus, the cre recombinase can be switched from an inducible form to a constitutively active form. Podocyte cellspecific expression of doxycycline inducible cre recombinase in mice. May 20, 2005 tcell ablation in cd4 cre idtr mice by application of dt. Transgenic mice carrying the human vitelliform macular dystrophy2 vmd2 promoter p vmd2directed reverse tetracyclinedependent transactivator rtta and the tetracyclineresponsive element tredirected cre were generated.
H mice, the thy1 promoter drives robust and relatively uniform expression of a drug. A creinducible diphtheria toxin receptor mediates cell. Tamoxifeninduced creloxp recombination is prolonged in. Cre recombinaseinducible rna interference mediated by. Conditional and inducible transgene expression in mice. 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. Verma, march 29, 2004 recently, several systems designed to trigger rna. Carbonic anhydrase i car1 is a gene expressed by colonic epithelial cells. Inducible cre expression was achieved by feeding doxycycline to these mice and was characterized by using a cre activatable lacz reporter mouse strain. Cre activation at the precise time and in a specific cell. Although tamoxifen inducible mice under pcaggs plasmid, containing cytomegalovirus enhancer chicken actin promoter and polyadenylation signal, have been shown to direct gene recombination in podocytes and other tissues 9, the podocytespecific tamoxifen inducible cre recombinase mice.
Basic cre lox mechanism strain types and breeding schemes o tissuespecific knockouts o general knockouts o inducible knockouts o reporters cre lox web resources finding mice, cre. Springer nature is developing a new tool to find and evaluate protocols. Pdf the crelox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. The use of the cre lox system involves both mice expressing cre enzyme and mice with loxp sites inserted at. These transgenic mice have a tamoxifen inducible cre mediated recombination system driven by the mouse plp1, proteolipid protein myelin 1 promoter.
Although tamoxifen inducible mice under pcaggs plasmid, containing cytomegalovirus enhancer chicken actin promoter and polyadenylation signal, have been shown to direct gene recombination in podocytes and other tissues 9, the podocytespecific tamoxifen inducible cre recombinase mice was not constructed. They provide trophic support for neurons, modulate synaptic transmission and plasticity, and contribute to neuronal dysfunction. Mhc extensively used to drive cardiomyocyterestricted recombination of floxed genes in animal models. Highly tamoxifeninducible principalcellspecific cre. The crelox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. Toassess the efficiency of induced cre mediateddeletion ofa target gene, wegenerated mice transgenic for a mxcreexpression vector 8 andcrossed themto a strain.
The genotype of each mouse with respect to the creert2 transgene and the rosa26 reporter gene is indicated in the boxes. Hence, mice harboring a floxed target gene and a mx cre transgene acquire an inactivating mutation of the target gene upon treatment of the mice with ifn or pipc. When crossed with a strain containing a loxp site flanked sequence of interest, the offspring are useful for generating tamoxifeninduced, cre mediated targeted deletions. 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. Highperformance chemical and lightinducible recombinases. The cre loxp system is widely used to excise the gene segment flanked by loxp recognition sequences floxed 10. Three expressing transgenic lines were tested and the two that expressed at higher levels gave the best, and essentially identical, results. In order to achieve temporally controlled somatic mutagenesis, the cre loxprecombination system has been complemented with the tamoxifen inducible fusion protein. Cre recombinaseinducible rna interference mediated by lentiviral vectors gustavo tiscornia, vinay tergaonkar, francesco galimi, and inder m.
Efficiency and specificity of gene deletion in lung. To study the roles of key transcription factor networks, growth factors, and signaling molecules in the endoderm, notochord, and floorplate, we developed an inducible cre expressing system for altering gene function in this tissue. Pdf system for tamoxifeninducible expression of cre. A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. It should be noted that some cells in the deep zone of the articular. Furthermore, transgenic mice bearing a ta moxifendependent cre recombinase vilcreert2 ex pressed under the control of the villin promoter were. Modification of gene activity in mouse embryos in utero by. Cardiacspecific inducible and conditional gene targeting in mice. C31 systems we developed along with crecontrolled interferon.
Dec 11, 2019 astrocytes are the most abundant cell type in the central nervous system cns. It allows the dna modification to be targeted to a specific cell type or be triggered by a specific external stimulus. Modification of gene activity in mouse embryos in utero by a. Inducible gene inactivation in neurons of the adult mouse. Mhcmercremer mice, cre recombinase is fused to two mutated estrogen receptor domains. Here, cre was used to delete a segment of the dna polymerase beta gene flanked by ioxp recombinase recognition sites. Methodology open access inducible cre transgenic mouse strain for skeletal musclespecific gene targeting john j mccarthy1,2, ratchakrit srikuea2,5, tyler j kirby3, charlotte a peterson1,4 and karyn a esser1,2,6 abstract background. Bac transgenesis to generate a mouse line expressing a tamoxifeninducible cre under the control of a remakexpressed gene promoter. Similar cre er fusion proteins have been reported by others, which can function in a liganddependent manner in adult mice.
Moon1,4,5 to study the roles of key transcription factor. Crelox basics generating knockout mice webinar 17mar2016. Lowell department of laboratory medicine and program in immunology, university of california, san francisco, california orcid id. However, 9 days after ifn induction, a population of igh b only b cells can be seen in lymph nodes figure 4, spleen, and peritoneal cavity of b1. Podocytespecific expression of tamoxifeninducible cre. Similar creer fusion proteins have been reported by others, which can function in a liganddependent manner in adult mice. Tamoxifen tm inducible cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Regulating rtta and target expression with cre teton krasg12d to model pancreatic cancer b6. Specificity and leakiness of inducible creloxp systems. Podocyte cellspecific expression of doxycycline inducible. 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.
Another technical concern regarding the use of cre mice is that these mice either can exhibit phenotypic abnormalities and. In addition, transgenic or knockin mice expressing tetoffegfpcre and egfpcreert2 driven by the promoters of foxd1 8, six2 9, and slc34a1 10, respectively, are also available. Before induction of cre expression, the majority of the b cells in b18fgld42i, mx cre mice coexpress both igh proteins, similar to control b18fgld42i mice depicted for pbl in figure 4. It is implemented both in eukaryotic and prokaryotic. However, many complete and tissuespecific cremediated knockout mice result in embryonic or early perinatal death, thereby precluding the analysis of gene. Oct 24, 2019 in this system, we utilize highperforming abainducible cre, gibinducible flp, and rapinducible. Conditional alkbh4 ll flanked by loxp mice crossed with creesr transgenic mice to create the inducible knockout genotype alkbh4 ll creesr overcoming the embryonic lethality. Stifter and melanie greter institute of experimental immunology, university of zurich, winterthurerstrasse 190, 8057, zurich, switzerland cre and creer mouse strains are powerful tools that have proven invaluable for investigating the function of genes and for the fatemapping of cell populations. Tcell ablation in cd4 cre idtr mice by application of dt. Eightyfive percent and 82% cre recombination efficiency were achieved in articular cartilage area of these mice fig. Mouse strains to study coldinducible beige progenitors. Deletion was complete in liver and nearly complete in lymphocytes within a. System for tamoxifeninducible expression of crerecombinase from the foxa2 locus in mice eon joo park,1 xiaoxia sun,2 peter nichol,3 yukio saijoh,1,4 james f.
Nih public access transgenic mice nat neurosci single. Basic cre lox mechanism strain types and breeding schemes o tissuespecific knockouts o general knockouts o inducible knockouts o reporters cre lox web resources finding mice, cre activity data, and more 7. Expression patterns of inducible cre recombinase driven by. Transgenic mice, inducible gene knockout, somatic mutagenesis. Methodology open access inducible cre transgenic mouse strain.
Inducible activation of cre recombinase in adult mice causes. Inducible activation of cre recombinase in adult mice. Upon crossing these mice to cre reporter strains e. Chapter 18 inducible cre mice susanne feil, nadejda valtcheva, and robert feil abstract the cre lox sitespecific recombination system has emerged as an important tool for the generation of. Strain types and breeding schemes o tissuespecific knockouts o general knockouts o inducible knockouts o reporters. Many of these inducible cre lines are active in multiple segments, particularly in proximal tubules. 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. Crelox technology and breeding schemes for research. The use of the cre loxp system for gene targeting has been proven to be a powerful tool for.
The results showed that tm induced efficient cre recombination in articular chondrocytes 1 and 6 months after tm induction fig. Investigate the effect of alkbh4 deficiency in a physiological context, using inducible alkbh4 knockout mice. By crossing with mice expressing cre under the control of tissuespecific or inducible promoters, cre mediated recombination will be. Cggtrosa26sortm1rtta,egfpnagyj 005670 dow 2014 plos one pmid. Tamoxifen inducible cre activity is observed in the developing kidney, head, ear and limb, mimicking the endogenous six2 expression pattern. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. Generation of nsemercremer transgenic mice with tamoxifen. Many transgenic mouse lines have been generated to obtain astrocytespecific expression of inducible cre recombinase for functional studies. We generated an inducible car1 creer mouse model with cre expression in the cecum and. Generation of an inducible colonspecific cre enzyme mouse. The commonly used recombinase, cre, recognizes specific sequences loxp, and catalyzes recombination between two loxp sites. An inducible cre system is controlled by cellspecific regulatory elements promoters and enhancers and temporally inducible way by exogenous inducer such as tamoxifen tam or tetracycline tet 1,10,1417. Highly tamoxifeninducible principal cellspecific cre. 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.
An inducible cre mouse line to sparsely target nervous. R26r rfp mice hm were administered one dose of tm on 2 consecutive days or administered dox 3 days. Transgenic construct nsemercremer was constructed by linking the rat nse promoter 5. An inducible podocytespecific gene expression has been demonstrated previously, and this inducible procedure has been used to construct inducible knockout mice.
Deletion was complete in liver and nearly complete in lymphocytes. Inducible activation of cre recombinase in adult mice causes gastric epithelial atrophy, metaplasia, and regenerative changes in the absence of. Inducible knockout of mouse zfhx3 emphasizes its key role. To investigate the functional properties of the idtr system in vivo, we crossed idtr mice to cd4 cre mice 31 in which the recombinase is. Transgenic mice inducible gene knockout somatic mutagenesis creer recombinase tamoxifen in utero rosa26 r26r xgal staining mouse models of human disease. Such mice have successfully been used to generate cre loxpmediated gene disruption in an inducible manner in the myocardium in vivo. Rapid and highly efficient inducible cardiac gene knockout. However, information is sparse regarding the tamoxifen dosage, the time course of gene disruption and whether different administration routes differ in efficiency in obtaining gene disruption in the myocardium.
Rapid and highly efficient inducible cardiac gene knockout in. 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. Transgenic mice, inducible gene knockout, somatic mutagenesis, creer recombinase, tamoxifen, in utero, rosa26, r26r, xgal staining, mouse. 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 assess. Cardiacspecific inducible and conditional gene targeting. Discussion in this study, we demonstrate that the patterns of targeted cells in inducible col2creer mice when tamoxifen is administered postnatally are partially overlapping with but different from the targeted cells in col2 cre mice. Inducible expression of cre recombinase in the retinal. Research article highly tamoxifen inducible principal cellspeci. Tamoxifen administration routes and dosage for inducible. Pdf the cre lox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. It is implemented both in eukaryotic and prokaryotic systems. A strategy for rapid screening of inducible cre constructs. Singleneuron labeling with inducible cre mediated knockout in transgenic mice paul young1,4, li qiu1, dongqing wang1, shengli zhao1, james gross1,3, and guoping feng1,2 1department of neurobiology, duke university medical center, research drive, durham, north carolina 27710, usa 2department of pathology, duke university medical center, research drive, durham, north carolina 27710, usa 3duke. The cre er tm cdna was placed under the regulation of the wnt1 enhancer.
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