Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells

Mahonen, Anitta; Airenne, Kari J.; Purola, Sanna; Peltomaa, Erik; Kaikkonen, Minna U.; Riekkinen, Mervi S.; Heikura, Tommi; Kinnunen, Kati; Roschier, Miia M.; Wirth, Thomas; Ylä‐Herttuala, Seppo

doi:10.1016/j.jbiotec.2007.05.022

Cited by 50 publications

(46 citation statements)

References 49 publications

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“…In general, improvements in transient gene expression are multiplex with the most prominent to be the transfection efficiency, the maximum cell density and the concentration of protein enhancers (Jardin et al 2008). Other enhancers are sodium butyrate (Leisy et al 2003;Mahonen et al 2007;Ping et al 2006) and trichostatin A (Spenger et al 2004). More recently proto-oncogenes, cell cycle control genes, growth factor genes and anti-apoptotic genes have been used to improve production hosts or transient expression (Wurm 2004;Backliwal et al 2008).…”

Section: Discussionmentioning

confidence: 99%

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

et al. 2009

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The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein.Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.

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Section: Discussionmentioning

confidence: 99%

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

et al. 2009

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“…Baculovirus Ba-CAG-EGFP, with woodchuck hepatitis posttranscriptional regulatory element (WPRE), and baculovirus p24Cherry, without WPRE, were used for the binding, entry, and transduction experiments. Sucrose gradient-purified viruses were produced as described earlier (16,43). Different multiplicities of infection (MOIs; 200, 400, 500, and 800) were used depending on experimental setup.…”

Section: Methodsmentioning

confidence: 99%

6-O- andN-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

Makkonen

Turkki

Laakkonen

et al. 2013

J Virol

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“…The WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), a cis-acting element that enhanced the baculovirus-mediated transgene expression in vertebrate cells, 46 was amplified from pFastBac1-CMV-EGFP-WPRE 30 (kindly provided by Professor Shu Wang of National University of Singapore) and inserted into pBac-hEA between the hEA gene and pA to yield pBac-hEA/w. The IR/ DR elements from pT2-BH were digested by SalI/NotI and cloned into pFastBacDpolhDp10 to yield pBac-T2.…”

Section: Preparation Of Recombinant Baculovirusesmentioning

confidence: 99%

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

et al. 2011

Gene Ther

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Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (o7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/ Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

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Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells

Cited by 50 publications

References 49 publications

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

6-O- andN-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

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