Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 °C

Beer, Christiane; Buhr, Petra; Hahn, Heidi; Laubner, Daniela; Wirth, Manfred

doi:10.1099/vir.0.18871-0

Cited by 30 publications

(23 citation statements)

References 30 publications

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“…Cells were maintained in Roswell Park Memorial Institute (RPMI 1640; Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and seeded the day before transfection at a density of 5 ϫ 10 4 cells per well in 12-well plates. Cells were then transfected with 900 ng of the reporter plasmid p11xGli1 or the empty control pGL3-TK (Promega, Madison, WI) 20 and 100 ng of the reference plasmid pRL-TK using FuGene 6 transfection reagent (Roche Diagnostics). Forty-eight hours after transfection, cells were lysed and reporter gene activity was determined using the Dual-Glo Luciferase Reporter Assay System (Promega).…”

Section: Methodsmentioning

confidence: 99%

Blocking the hedgehog pathway inhibits hepatoblastoma growth

Eichenmüller¹,

Gruner²,

Hagl³

et al. 2008

Hepatology

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Recent evidence has indicated that Hedgehog (Hh) signaling significantly contributes to liver development and regeneration and that activation of the pathway may contribute to growth of hepatocellular carcinoma (HCC) in adults. However, the role of Hh signaling in pediatric liver tumors remains to be elucidated. In this study, we show that Hh signaling is activated in hepatoblastoma (HB), the most common liver tumor in childhood, with most occurrences before the age of 3 years. The Hh target genes glioma-associated oncogene homolog 1 (GLI1) and Patched (PTCH1) showed increased transcript levels in 65% and 30% of HB samples, respectively, compared with normal liver tissues. Most interestingly, the gene encoding the hedgehog interacting protein (

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Section: Methodsmentioning

confidence: 99%

Blocking the hedgehog pathway inhibits hepatoblastoma growth

Eichenmüller¹,

Gruner²,

Hagl³

et al. 2008

Hepatology

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“…In recent years there has been an ever-growing number of reports that the sub-physiological temperature in vitro culturing (<37°C) of mammalian cells can lead to enhanced heterologous protein production (Beer et al 2003;Ducommun et al 2002;Fogolin et al 2004;Fox et al 2004;Fox et al 2005b;Schatz et al 2003;Yoon et al 2003a;Yoon et al 2003b). This effect is extremely variable however, and appears to be both cell line and expression system dependent .…”

Section: Introductionmentioning

confidence: 99%

The cold-shock response in mammalian cells: investigating the HeLa cell cold-shock proteome

Underhill

Smales

2007

Cytotechnology

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In recent years there have been a number of reports that suggest the sub-physiological (<37°C) temperature in vitro culturing of mammalian cells can result in enhanced heterologous protein production. Despite these reports, the mechanisms by which mammalian cells respond to such conditions are largely unknown. We therefore set out to use a model in vitro culture HeLa cell system to begin investigating the cold-shock response in mammalian cell systems. Sub-physiological temperature cultivation resulted in reduced growth and proliferation and a lower total cell protein content. Proteomic analysis confirmed that HeLa cells actively respond to sub-physiological temperature by upregulating a number of proteins and immunoblot analysis confirmed that specific proteins are indeed up-regulated in a time and temperature dependent manner. Additional work is likely to improve our understanding of the cold-shock response in mammalian cells and identify candidate target proteins for cell engineering to further enhance heterologous protein production at sub-physiological temperatures.

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“…6,7 Moreover, the cholesterol content enhances the viral membrane rigidity, which negatively affects infectivity of human immunodeficiency virus 24,25 and amphotropic MVL. 26 To improve vector quality, production methods are being designed to optimize cholesterol content in viral membranes, 27 and thereby improving vector stability. Retroviral vector characteristics may be manipulated through specific changes in the production temperature and medium composition (for example, with decreased cholesterol concentrations or cholesterol inhibitors).…”

Section: Discussionmentioning

confidence: 99%

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years

et al. 2008

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Recombinant retroviruses are one of the most commonly used gene transfer vehicles for therapeutic gene delivery. The stability of viral vectors upon long-term storage, anticipated to be short lived, is expected to impact timeline and financial course of clinical immunogene therapy. However, to date little is known about vector stability. Therefore, we analyzed the stability of retroviral vectors produced in culture supernatants (RTVsup) for ex vivo gene therapy upon long-term storage. We have generated RTVsups derived from two packaging cell lines, PG13 and Phoenix(Ampho). Both lines produced murine leukemia virus-derived SFG-scFv(G250)-CD4g vector, which were pseudotyped with the gibbon ape leukemia virus envelope and amphotropic envelope, respectively. The supernatants were stored at À80 or À196 1C. To date, the PG13-derived RTVsups have been evaluated over a period of 9 years (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007). In addition, a clinical batch of Phoenix(Ampho)-derived RTVsup has been evaluated over a period of 5 years (2002)(2003)(2004)(2005)(2006)(2007). Here, we show that both RTVsups, when stored up to 9 and 5 years, respectively, do not show any sign of decay in their capacity to functionally transduce primary human T cells. These data provide evidence that in terms of 'life expectancy' the production and storage of clinical batches of RTVsup for gene therapy warrants the corresponding professional and financial risks.

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Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 °C

Cited by 30 publications

References 30 publications

Blocking the hedgehog pathway inhibits hepatoblastoma growth

Blocking the hedgehog pathway inhibits hepatoblastoma growth

The cold-shock response in mammalian cells: investigating the HeLa cell cold-shock proteome

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years

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