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Highly inducible cell lines derived from mice genetically transmitting the Moloney murine leukemia virus genome
Abstract:
Permanent, non-virus-producing cell lines have been established from a mouse embryo carrying an endogenous, genetically transmitted Moloney murine leukemia virus (M-MuLV) genome. These cells carry the M-MuLV genome, as demonstrated by hybridization of cellular DNA to M-MuLV complementary DNA, but do not express it at the levels of virus production, accumulation of intracellular viral p30, or M-MuLV-specific RNA. Treatment with bromodeoxyuridine (50 microgram/ml for 24 h) resulted in induction of XC-positive NB-tropic virus, although only a small fraction of the cells released virus (less than 0.1% after 48 h). Immunofluorescent staining and flow microfluorometry indicated that a wave of p30 accumulation occurs in the induced cells, with a maximum at 24 to 48 h after the addition of bromodeoxyuridine. Furthermore, most, if not all, cells were induced to produce p30 protein. Similar kinetics were found for the accumulation of M-MuLV-specific RNA in the cytoplasm of induced cells. This rapid induction of virus expression in a majority of cells was dependent on the presence of the M-MuLV genome and probably represents primarily the expression of this endogenous virus since induction was not observed in cells similarly derived from a sibling embryo lacking the M-MuLV genome.
Insights
Researchers established permanent cell lines from mice with an endogenous Moloney murine leukemia virus (M-MuLV) genome. Bromodeoxyuridine treatment induced M-MuLV expression, demonstrating efficient viral gene activation in these cells.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Endogenous retroviruses can be stably integrated into host genomes.
- Understanding the regulation of endogenous retroviral expression is crucial for virology research.
- Moloney murine leukemia virus (M-MuLV) is a well-studied retrovirus with implications for oncogenesis.
Purpose of the Study:
- To establish and characterize permanent cell lines from mice carrying an endogenous M-MuLV genome.
- To investigate the inducibility of M-MuLV expression in these cell lines.
- To analyze the molecular mechanisms underlying M-MuLV gene activation.
Main Methods:
- Establishment of permanent cell lines from M-MuLV-carrying mouse embryos.
- DNA-DNA hybridization to confirm M-MuLV genome presence.
- Treatment with bromodeoxyuridine (BrdU) to induce viral expression.
- XC plaque assays for virus production.
- Immunofluorescence staining and flow microfluorometry for p30 protein detection.
- Northern blot analysis for M-MuLV-specific RNA quantification.
Main Results:
- Permanent cell lines carrying the endogenous M-MuLV genome were successfully established.
- BrdU treatment induced XC-positive NB-tropic virus production in a small fraction of cells (<0.1%).
- A significant wave of p30 protein accumulation and M-MuLV-specific RNA was observed in most induced cells within 24-48 hours.
- Induction was dependent on the presence of the M-MuLV genome, confirming endogenous viral expression.
Conclusions:
- Permanent cell lines from M-MuLV-carrying embryos can be established and maintain the viral genome without producing infectious virus.
- Bromodeoxyuridine is an effective inducer of endogenous M-MuLV expression, leading to widespread p30 protein and RNA accumulation.
- These findings provide a valuable model system for studying the regulation of endogenous retroviral gene expression and activation.