pEF-BOS, a powerful mammalian expression vector

Mizushima, Seiichi; Nagata, Shinji

doi:10.1093/nar/18.17.5322

Cited by 1,579 publications

(907 citation statements)

References 4 publications

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“…The recombinant sCD200 protein was purified from spent tissue culture media on Ni columns (Qiagen, Hilden, Germany) and eluted with 250 mM imidazole in phosphate-buffered saline pH 8.0. The mutants of CD200RCD4d3+4biotin were prepared by overlapping PCR and cloned into pEFBOS [27] vector. The recombinant proteins were transiently expressed in 293T cells and assayed by ELISA [28].…”

Section: Expression Of Recombinant Scd200 and Cd200r Mutant Proteinsmentioning

confidence: 99%

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

Hatherley

Barclay

2004

Eur J Immunol

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CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) involved in regulation of macrophage function. Both CD200 and CD200R contain two Ig superfamily domains like many other leukocyte membrane proteins. Site‐directed mutagenesis of CD200R showed that, like CD200, it interacted through its N‐terminal domain. This indicated that the cell‐cell interaction spans four Ig superfamily domains and this distance is similar to many interactions found between T cells and antigen‐presenting cells. This suggests that this topology is also important in interactions of CD200 on a variety of cells with CD200R on myeloid cells, and comparable contact sites may be important mediating regulation in other cell‐cell interactions. The mutagenesis showed that the binding involved the predicted GFCC′ face of its N‐terminal domain, like that of CD200, suggesting that the interaction evolved from a homotypic interaction.

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Section: Expression Of Recombinant Scd200 and Cd200r Mutant Proteinsmentioning

confidence: 99%

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

Hatherley

Barclay

2004

Eur J Immunol

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“…The murine Ews/Fli-1 fusion cDNA construct was generated by first amplifying exons 1 -7 on murine Ews cDNA and exons 6 -9 of murine Fli-1 cDNA, and then fusing both partial cDNAs together by using an overlap extension PCR technique. The murine Ews/Fli-1 and human type 1 EWS/FLI-1 fusion genes were cloned into the pEF-BOS vector (Mizushima and Nagata, 1990) containing a puromycin resistance gene. Similar constructs were also generated using the cytomegalovirus (CMV) minimal promoter.…”

Section: Plasmidsmentioning

confidence: 99%

Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1

Chan

Wilson

et al. 2003

Br J Cancer

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Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

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“…The primary plasmid vector, pNAss (Clontech, Palo Alta, CA, USA), has a pUC19 backbone with an ampicillin-resistance gene, a CMV promoter, a SV40 splicing donor/splicing acceptor (SD/SA) sequence and an SV40 polyadenylation signal sequence. Two other plasmid expression vectors that were also evaluated were: the pWRG3147 vector contains an elongation factor (EF)-1a promoter, 33 the SV40 intron 1 sequence, and an SV40 polyadenylation sequence. The pWRG 7007 vector contains a CMV promoter followed by the CMV intron A enhancer sequence, a bovine growth hormone (bGH) polyadenylation signal sequence and a kanamycin-selection gene.…”

Section: Optimization Of Concomitant/coordinated Expression Of Two Sumentioning

confidence: 99%

“…The CMV promoter was purchased from Clontech, the CMV/intron A element was obtained from Drs J Arthoson and JI Mullins, University of Washington Health Sciences Center, Seattle, Washington 4 and the EF-1a promoter subcloned from an EF containing plasmid was provided by Dr Shigekazu Nagata, Osaka Bioscience Institute, Osaka, Japan. 33 Human IL-12 subunit p35 and p40 cDNA fragment genes were cloned from mRNA of a human lymphoblastoid cell line using a commercial RT-PCR kit (Gibco/BRL). Murine IL-12 subunits were cloned from mouse cDNA libraries as previously described.…”

Section: Optimization Of Concomitant/coordinated Expression Of Two Sumentioning

confidence: 99%

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

Turner

2003

Gene Ther

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The augmentation and optimization of specific targeted transgene expression systems are important strategies for clinical research into gene therapy and DNA vaccination, due to safety considerations. In this study, we introduced 3 0 untranslated regions and transcriptional control modifications and direct tandem or combinational vector design strategies into a number of specific cytokine cDNA expression plasmids. The experiments were performed in parallel using both in vivo and in vitro transgene expression systems. In vivo studies were carried out using gene gun delivery of test vectors into mouse skin tissues. A combination of specific cell lines and fresh cell explants were used for in vitro and ex vivo transgene expression assay systems. The results from these comparative experiments demonstrated that a number of molecular biology manipulations can be readily adapted to define and significantly enhance the level or/and duration of transgene expression for a group of clinically relevant cytokine genes, with very similar effects for both in vivo and in vitro test systems. This cytokine transgene expression system may offer a favorable means for improving the efficiency of cytokine gene therapy and DNA vaccines in future clinical studies.

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pEF-BOS, a powerful mammalian expression vector

Cited by 1,579 publications

References 4 publications

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

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