共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
4.
5.
6.
Yu SS Dan K Chono H Chatani E Mineno J Kato I 《Biochemical and biophysical research communications》2008,368(4):942-947
Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells. 相似文献
7.
8.
9.
10.
11.
In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors 总被引:9,自引:0,他引:9
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Ulrich Kuhn Atsushi Terunuma Wolfgang Pfutzner Ruth Ann Foster Jonathan C. Vogel 《Journal of virology》2002,76(3):1496-1504
For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. 相似文献
12.
Mechanism of action of regulatory proteins encoded by complex retroviruses. 总被引:50,自引:0,他引:50
下载免费PDF全文
![点击此处可从《Microbiological reviews》网站下载免费的PDF全文](/ch/ext_images/free.gif)
B R Cullen 《Microbiological reviews》1992,56(3):375-394
13.
14.
Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility 总被引:2,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Seon Hee Kim Seung Shin Yu Jong Sang Park Paul D. Robbins Chung Sun An Sunyoung Kim 《Journal of virology》1998,72(2):994-1004
Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy. 相似文献
15.
16.
Virus-mediated reprogramming of gene expression in plants 总被引:8,自引:0,他引:8
17.
18.
Retroviral gene transfer and bone marrow transplantation has been used by many investigators to study the role of macrophage
proteins in different mouse models of human disease. While this approach is faster and less expensive than generating transgenic
mice with macrophage-specific promoters and applicable to a wider array of mouse models, it has been hampered by two major
drawbacks: labor-intensive cloning procedures involved in generating retroviral vectors for each gene of interest and low
viral titers. Here we describe the construction of a MSCV-based retroviral vector that can serve as an acceptor vector for
commercially available Cre-lox-compatible donor vectors. Using this new retroviral vector in combination with a FACS approach
to enhance viral titers, we generated high-titer retroviruses carrying either EGFP-tagged cytosolic or EGFP-tagged mitochondria-targeted
glutathione reductase. We show that the introduction of these constructs via retroviral gene transfer and bone marrow transplantation
into atherosclerosis-prone LDL receptor-null mice results in the long-term increase in macrophage glutathione reductase activity. 相似文献
19.
The ability to transfer permanently genes into mammalian cells makes retroviruses suitable vectors for the ultimate purpose of treating inherited genetic disease. However, expression of the retrovirally transferred genes is variable (position effect and expression variegation) because retroviruses are highly susceptible to the influence of the host genome sequences which flank the integration site. We have investigated this phenomenon with respect to the human housekeeping enzyme, glucose 6-phosphate dehydrogenase (hG6PD). We have constructed retroviral vectors in which the hG6PD cDNA is driven by either of two conventional retroviral promoters and enhancers from the Moloney Murine Leukemia Virus (MMLV) and the Myeloproliferative Sarcoma Virus (MPSV) long terminal repeats (LTR) or by the hG6PD own promoter replacing most of enhancer and promoter LTR (GRU5). We have compared the activity of retrovirally transferred hG6PD driven by these promoters after retroviral integration in bulk cultures and in individual clones of murine fibroblasts. The level of hG6PD expressed by the hG6PD promoter of GRU5-G6PD was significantly lower than that expressed by conventional retroviral vectors. However, analysis of the single copy clones showed less variation of expression with GRU5-G6PD (coefficient of variation, CV, 35.5%) than with conventional vectors (CV, 58.9%). Thus we have several vectors competent for reliable transfer and expression of hG6PD. The hG6PD promoter provides reproducible expression of hG6PD and limits the variability of expression. This decreased variability is important in order to help ensuring a consistent level of delivery of the needed gene product in future therapeutic protocols. 相似文献
20.
Alice T Trinh Bret G Ball Erin Weber Timothy K Gallaher Zoya Gluzman-Poltorak French Anderson Lena A Basile 《Genetic vaccines and therapy》2009,7(1):1-12