首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到10条相似文献,搜索用时 78 毫秒
1.
Sequence requirements for plasmid nuclear import   总被引:8,自引:0,他引:8  
The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.  相似文献   

2.
3.
A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line. First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human beta-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media. Southern blot analysis revealed that the beta-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells. Next, the t-PA expression plasmid containing the beta-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines. Some of the transfectants from both cell lines were also found to produce t-PA. It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5.  相似文献   

4.
5.
The gene for Menkes disease codes for a Cu-transporting ATPase that regulates Cu homeostasis in all tissues with the exception of adult liver. The basis for developmental or tissue-specific regulation at present is not understood. To learn if the regulation is associated with the promoter, we cloned and sequenced a 2.2 kb genomic DNA fragment flanking exon 1. When ligated into a pGL2 luciferase reporter gene construct, the 2.2 kb showed promoter activity, but not nearly to the extent of a 1.3 kb fragment previously reporter by Levinson et al. Sequence analysis of the nucleotides spanning the region between 1.3 kb and 2.2 kb revealed a 13-nucleotide motif ACACAAAAAAATA 2059 bp upstream from the start site that duplicated the `hunchback' binding site, a key site controlling developmental gene expression in Drosophila. Eliminating 129 bp containing the hunchback site (Hb) from the 5 end of the 2.2 kb stimulated promoter activity, suggesting the Hb site was basically suppressive. When ligated upstream of an SV40 and tested in SY5Y cells, however, the SV40 promoter activity was strongly stimulated, which conflicts with the site being suppressive. Mutating the site in the 2.2 kb weakened the promoter activity in SY5Y and HepG2 cells and a fragment with mutated sequence ligated upstream of the SV40 cancelled the activation of SV40 promoter activity. All data suggested the Hb site was a positive controlling site for Cu-ATPase expression. Nuclear extracts from SY5Y and HepG2 cells were observed to bind to a 106 bp probe with the Hb site in a gel-shift assay. Only SY5Y proteins, however, showed a slower moving shift band indicative of a secondary interaction. A probe with mutated sequences displayed the same shift pattern, suggesting other sites in the 106 bp DNA strand were also recognizing the nuclear proteins. A Southwestern analysis suggested that proteins of 125 kD, 70 kD, 50 kD and 42 kD bound to the wild type probe; a 60 kD and all with the exception of the 42 kD bound to the mutant probe. The data support the conclusion that the distal promoter of the Menkes disease gene contains elements that interact in combinatorial fashion to regulate Cu-ATPase expression and that tissue specificity may lie with the quantity or types of distinct DNA binding proteins in the nucleus.  相似文献   

6.
7.
Yue Y  Dongsheng D 《BioTechniques》2002,33(3):672, 674, 676-672, 674, 678
Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient.  相似文献   

8.
Summary The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.  相似文献   

9.
10.
Q vectors, bicistronic retroviral vectors for gene transfer   总被引:3,自引:0,他引:3  
We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号