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1.
Lundstrom K 《Journal of receptor and signal transduction research》2002,22(1-4):229-240
Semliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture. Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification. The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies. Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range. 相似文献
2.
《Journal of receptor and signal transduction research》2013,33(1-4):229-240
ABSTRACTSemliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture. Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification. The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies. Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range. 相似文献
3.
Semliki Forest virus vectors (SFV) have been developed for efficient transgene expression to result in high receptor yields(50–200
pmol receptor/mg protein) in a variety of mammalian host cells. Transfer of the SFV technology to mammalian cells growing
in suspension cultures has made it feasible to produce hundreds of milligrams of receptor proteins in a short time. Large-scale
production, however, raises the questions of the safety of handling virally infected cells for down-stream processing. Analysis
of cell culture medium and SFV-infected cells revealed that some infectious particles were still present. Replacement of virus-containing
medium at 2 h post-infection efficiently removed the majority of infectious replication-deficient SFV particles. Washes with
PBS further reduced the number of infectious particles significantly both in the medium and associated with cells to levels
that allowed safe handling of SFV-infected cells outside the cell culture facility for biochemical, pharmacological, or electrophysiological
assays or down-stream processes in connection to receptor purification. Furthermore, engineering of novel temperature-sensitive
mutant SFV vectors resulted in temperature-controlled transgene expression, which completely eliminates the risk of contaminating
laboratory personnel.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
4.
Ehrengruber MU 《Molecular neurobiology》2002,26(2-3):183-201
Alphaviruses are small, enveloped positive-strand RNA viruses that have been successfully transformed into expression vectors
in the case of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus. Compared to other
viral vectors, their advantages are easy and fast generation of recombinant viral particles, rapid onset, and high-level transgene
expression. When applied to neuronal tissue, SFV and SIN vectors possess the additional advantage of efficiently and preferentially
transducing neurons rather than non-neuronal cells. This article gives an overview of the biology of SFV and SIN, their generation
into expression vectors, and their application in neurobiology, with particular emphasis on the transduction of hippocampal
neurons. In addition, it describes the more recent development of alphaviral vectors with decreased or absent cytotoxicity
and lowered transgene expression, temperature-controllable gene expression, and altered host-cell specificity in the central
nervous system (CNS). Finally, the review evaluates the use of SFV and SIN vectors in hippocampal tissue cultures vs recombinant
lentivirus, adenovirus type 5, adeno-associated virus type 2, and measles virus. 相似文献
5.
Novel mutant Semliki Forest virus vectors: gene expression and localization studies in neuronal cells 总被引:3,自引:0,他引:3
Lundstrom K Rotmann D Hermann D Schneider EM Ehrengruber MU 《Histochemistry and cell biology》2001,115(1):83-91
Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology. 相似文献
6.
The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles
generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines
growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant
protein expression. For example, the luciferase activity was significantly higher in the rodent BHK and CHO cell lines incubated
at 33 °C compared to 37 °C when harvested 19 h post-infection. At 33 °C the specific expression levels increased 10–20 fold
during prolongation of the post-infection time up to 50 h. In contrast, a significant decrease in luciferase activity was
observed from 26 h post-infection for cell cultures incubated at 37 °C. Only a slight temperature effect on luciferase expression
was seen in the human cell line HEK293 and no effect was observed for the subclone293(EBNA).
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
7.
Jannik E. Jakobsen Juan Li Brian Moldt Peter M. Kragh Henrik Callesen Jens Michael Hertz Lars Bolund Arne Lund Jørgensen Jacob Giehm Mikkelsen Anders Lade Nielsen 《Molecular biology reports》2011,38(1):151-161
We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed
transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with
a Sleeping Beauty DNA transposon-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase
site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and
generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear transfer. PFV cells supported
Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated
blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell
lines from other mammalian species suitable for locus-directed transgene expression in cell cultures and, in addition, for
transgene analyses in the very early embryonic stages. 相似文献
8.
Gas-inducible transgene expression in mammalian cells and mice 总被引:7,自引:0,他引:7
Weber W Rimann M Spielmann M Keller B Daoud-El Baba M Aubel D Weber CC Fussenegger M 《Nature biotechnology》2004,22(11):1440-1444
9.
Lundstrom K 《Journal of receptor and signal transduction research》1999,19(1-4):673-686
The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80-95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing beta-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells. 相似文献
10.
《Journal of receptor and signal transduction research》2013,33(1):55-70
We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in β-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications. 相似文献
11.
K Lundstrom P Ziltener D Hermann C Schweitzer J G Richards F Jenck 《Journal of receptor and signal transduction research》2001,21(1):55-70
We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in beta-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications. 相似文献
12.
《Journal of receptor and signal transduction research》2013,33(1-4):673-686
AbstractThe broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80–95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing β-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells. 相似文献
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15.
Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins 总被引:1,自引:0,他引:1
Lundstrom K 《Biochimica et biophysica acta》2003,1610(1):90-96
Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs. 相似文献
16.
Sen S Jaakola VP Heimo H Engström M Larjomaa P Scheinin M Lundstrom K Goldman A 《Protein expression and purification》2003,32(2):265-275
The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology. 相似文献
17.
Kenneth Lundstrom 《生物化学与生物物理学报:生物膜》2003,1610(1):90-96
Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs. 相似文献
18.