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Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. 总被引:13,自引:1,他引:12
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A K Indra X Warot J Brocard J M Bornert J H Xiao P Chambon D Metzger 《Nucleic acids research》1999,27(22):4324-4327
Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T). 相似文献
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We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis. 相似文献
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The study of gene functions in complex genetic environments such as mammalian cells would greatly benefit from systems allowing a tight control of gene expression. The tetracycline-inducible gene expression system and the site-specific Cre/loxP recombination system have gained increasing popularity for conditional expression and gene disruption. To facilitate the analysis of gene functions in a cell autonomous system, we have established an F9 murine embryonal carcinoma cell line, constitutively expressing both the doxycycline-controlled transactivator rtTA and the tamoxifen-dependent Cre recombinase Cre-ER(T). The expression of a reporter gene placed under the control of tetracycline operators was induced about 1000-fold by doxycycline, and tamoxifen-induced excision of a loxP-flanked DNA segment occurred in all cells. This genetically engineered cell line, which allows, upon simple ligand addition, sophisticated genetic manipulations, such as sequential inactivation of loxP-flanked genes, and tightly controlled reexpression of their cDNAs, should be a valuable tool for studying mammalian gene functions. 相似文献
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To generate temporally controlled targeted somatic mutations selectively and efficiently in skeletal muscles, we established a transgenic HSA-Cre-ER(T2) mouse line in which the expression of the tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the human skeletal muscle alpha-actin gene, contained in a P1-derived artificial chromosome. In this transgenic line Cre-ER(T2) is selectively expressed in skeletal muscles, and Cre-ER(T2)-mediated alteration of LoxP flanked (floxed) target genes is skeletal muscle-specific and strictly tamoxifen-dependent. HSA-Cre-ER(T2) mice should be of great value to analyze gene function in skeletal muscles, and to establish animal models of human skeletal muscle disorders. 相似文献
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Yuichi Iida Jung-Hyun Kim Yasuhiro Kazuki Hidetoshi Hoshiya Masato Takiguchi Masahiro Hayashi Indri Erliandri Hee-Sheung Lee Alex Samoshkin Hiroshi Masumoto William C. Earnshaw Natalay Kouprina Vladimir Larionov Mitsuo Oshimura 《DNA research》2010,17(5):293-301
Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoidtet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy. 相似文献
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Site- and time-specific gene targeting in the mouse 总被引:25,自引:0,他引:25
The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting. 相似文献
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RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking. 相似文献
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Developmental control genes are often sequentially and repeatedly functional during embryogenesis, and for this reason conditional mutagenesis tools are often required to study their roles in detail. Cre recombinase fused to the modified estrogen hormone-binding domain (ER(Tm)) generates a Cre in which the recombination activity of the LoxP-containing gene can be regulated by the nonsteroidal estrogen analogue 4-hydroxytamoxifen (4OH-TM). ER(Tm) may provide a useful way of achieving conditional mutagenesis in conjunction with the classic organ culture methods of experimental embryology. We used embryonic kidneys separated from the Cre-ER(Tm); R26R embryos to assay whether efficient 4OH-TM-inducible genomic recombination can be achieved in organ culture and in experimentally induced kidney mesenchymes. Our results indicate that the inducible ER(Tm) Cre/loxP system indeed provides an effective way of conditionally mutagenizing genes in kidney organ culture and tissue conjugates. 相似文献
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Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement. 相似文献
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Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system. 相似文献
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Bacteriophage T7 RNA polymerase-directed, inducible and tissue-specific over-expression of foreign genes in transgenic plants 总被引:1,自引:0,他引:1
A widely applicable bacteriophage T7 RNA polymerase-directed, tissue-specific and inducible over-expression of foreign genes in transgenic plants was developed. This was achieved through the simultaneous transformation of a modified T7 RNA polymerase to specifically transcribe the foreign gene placed under the control of T7 expression signals. The T7 RNA polymerase recognized the chimeric uidA gene integrated randomly into tobacco and rice genomes. Results from the use of six different promoters with different tissue specificities indicated that the recombinant protein was expressed at a several-fold (3-10-fold) higher level when compared with transgenes expressed directly under the control of these tissue-specific promoters. An important feature of the T7 system in plants was the near-uniform expression in the independently transformed plants, in contrast with the large variations observed in transgene expression under the direct control of plant promoters. In addition, our results demonstrated the application of the T7 system in the regulation of transgene expression through chemically inducible mechanisms. This versatility of controlled and regulated expression offers a powerful tool that could be used in various programmes in plant biotechnology and genomic studies. 相似文献
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A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages. 相似文献
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Uno S Wang B Shertzer HG Nebert DW Dalton TP 《Biochemical and biophysical research communications》2003,312(2):494-499
To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase. When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence-creating a conditional knockout allele. Targeted conditional alleles contain a minimum of three lox sites. It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene). Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome p450 1A1 (Cyp1a1) targeted allele. Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene. Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele. 相似文献
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Tchorz JS Suply T Ksiazek I Giachino C Cloëtta D Danzer CP Doll T Isken A Lemaistre M Taylor V Bettler B Kinzel B Mueller M 《PloS one》2012,7(1):e30011
Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice. 相似文献