共查询到20条相似文献,搜索用时 453 毫秒
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Replication of bovine papillomavirus vectors in murine cells 总被引:2,自引:0,他引:2
Margareta Waldenstrm Kerstin Schenstrm Kerstin Sollerbrant Lennart Hansson 《Gene》1992,120(2):175-181
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Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events
Pedro H. Oliveira Kristala Jones Prather Duarte M. F. Prazeres Gabriel A. Monteiro 《Applied microbiology and biotechnology》2010,87(6):2157-2167
Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine
development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical
to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high
recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and
mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison
to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting
a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters
and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp
direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding
is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because
pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped
several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential
targets to be removed. 相似文献
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Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells 总被引:7,自引:1,他引:6
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Hematti P Hong BK Ferguson C Adler R Hanawa H Sellers S Holt IE Eckfeldt CE Sharma Y Schmidt M von Kalle C Persons DA Billings EM Verfaillie CM Nienhuis AW Wolfsberg TG Dunbar CE Calmels B 《PLoS biology》2004,2(12):e423
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Mamuka Kvaratskhelia Amit Sharma Ross C. Larue Erik Serrao Alan Engelman 《Nucleic acids research》2014,42(16):10209-10225
Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. 相似文献
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《Gene》1998,222(2):305-318
Two cis regulatory elements of the human CD34 gene, the promoter and a 3′ enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3′ enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3′ enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression. 相似文献
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Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. 总被引:4,自引:3,他引:4
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The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter. 相似文献
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Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes 总被引:6,自引:0,他引:6
Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation. 相似文献