Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

May, Tobias; Lindenmaier, Werner; Wirth, Dagmar; Mueller, Peter P.

doi:10.1007/s10616-005-2834-z

Cited by 6 publications

(5 citation statements)

References 53 publications

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“…Fibroblasts were obtained from the skin of C57Bl/6J mice following the protocol as previously described [42] . Bone marrow derived DCs were generated from AEP −/− , CatB −/− , CatK −/− , CatL −/− , CatS −/− , TLR7 −/− mice and their wt littermates as previously described [16] .…”

Section: Methodsmentioning

confidence: 99%

Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation

et al. 2012

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Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.

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Section: Methodsmentioning

confidence: 99%

Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation

et al. 2012

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“…To our knowledge, an MCMV-permissive LSEC line is not available. Transduction of doxycycline-controlled expression cassettes encoding immortalizing genes such as SV40 T antigen can conditionally immortalize murine cells [22,23]. Cell lines established with this protocol express T antigen in a doxycycline-dependent fashion, allowing the control of the cell cycle and proliferation.…”

Section: Resultsmentioning

confidence: 99%

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

Dağ

Weingärtner

Butueva

et al. 2013

Virol J

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BackgroundThe MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood.MethodsWe constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells.ResultsMCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells.ConclusionBy means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

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“…In this study, the overall increase in production was 2-fold higher in the growth-arrested cells. Importantly, this ratio remained constant throughout the cultivation period [92].…”

Section: Controlled Cell Proliferation In Protein Production Processesmentioning

confidence: 88%

2.1 Generation of Cell Lines and Biotechnological Applications

Lipps¹,

May²,

Wirth³

2014

Animal Cell Biotechnology

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Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

Cited by 6 publications

References 53 publications

Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation

Asparagine Endopeptidase Controls Anti-Influenza Virus Immune Responses through TLR7 Activation

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

2.1 Generation of Cell Lines and Biotechnological Applications

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