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1.
The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.  相似文献   

2.
The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre-mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.  相似文献   

3.
Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.  相似文献   

4.
For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.  相似文献   

5.
Summary: The neuron‐specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre‐mediated transgene activation was determined by PCR and/or X‐gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE‐Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole‐mount X‐gal staining of 9.5 dpc embryos indicated Cre‐mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5–13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre‐mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE‐Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain. genesis 31:118–125, 2001. © 2001 Wiley‐Liss, Inc.  相似文献   

6.
利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.  相似文献   

7.
Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.  相似文献   

8.
组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性条件敲除研究的关键。采用PCR扩增大鼠胰岛素基因705bp启动子指导发胰岛细胞中特异表达;同时采用改构的Cre重组酶基因,在其5'端添加有真核核糖体结合序列和核定位序列使Cre重组酶能穿越核膜在细胞核能发挥功能;同时,为了保证原核基因Cre能在真核系统顺利表达,在其3'端添加含内含子的人生长激素基因。构建的表达载体在去除原核序列后用显微注射方法转基因小鼠,在出生的27只仔鼠中,PCR检测共获得7只Cre整合阳性的转基因小鼠,整合率26%。这种Cre转基因小鼠与基因组小携带LoxP位点的条件基因打靶小鼠交配,在胰腺组织中可以检测到Cre介导的重组,表明Cre在转基因小鼠胰腺中有表达。  相似文献   

9.
10.
胰腺组织表达Cre重组酶转基因小鼠的建立及鉴定   总被引:16,自引:0,他引:16  
组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性基因剔除研究的重要工具。为了建立胰腺组织特异性Cre转基因小鼠,我们通过PCR克隆了大鼠胰岛素基因启动子,并用它指导Cre基因在胰岛细胞中的特异性表达。在Cre重组酶基因5′端添加了真核核糖体结合序列和核定位序列以使Cre重组酶能穿越核膜在细胞核中发挥功能;同时,在Cre基因3′端添加了含内含子的3′端人生长激素基因。表达载体经显微注射导入小鼠受精卵以建立转基因小鼠。PCR检测显示共获得7只Cre整合阳性的转基因首建者小鼠;RTPCR结果表明其中1只首建者小鼠的子代鼠在胰腺中转录了外源基因,进一步的Southern杂交结果表明,该转基因小鼠能够在胰腺中表达有功能的Cre重组酶。   相似文献   

11.
12.
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   

13.
Cre-mediated gene deletion in the mammary gland.   总被引:22,自引:1,他引:21       下载免费PDF全文
To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.  相似文献   

14.
Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.  相似文献   

15.
16.
Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.  相似文献   

17.
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19.
The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.  相似文献   

20.
DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.  相似文献   

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