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Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor
Abstract:
Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.
Insights
Researchers explored the L factor, a multicopy mammalian plasmid, as a potential vector for gene delivery. Composite DNA constructs were successfully established in mouse cells, maintaining stability without selective pressure, indicating its promise for mammalian gene expression.
Area of Science:
- Molecular Biology
- Mammalian Cell Culture
- Gene Delivery Systems
Background:
- A multicopy mammalian plasmid, termed L factor, with structural similarities to polyoma, was previously identified at high copy numbers (≥5,000 per cell) in a mouse L cell subclone.
- The potential of this naturally occurring high-copy-number plasmid as a vector for mammalian cells was unexplored.
Purpose of the Study:
- To investigate the utility of the L factor plasmid as a vector for gene delivery in mammalian cells.
- To construct and evaluate composite DNA molecules comprising L factor and a foreign gene (herpes simplex virus thymidine kinase).
Main Methods:
- Construction of composite DNA molecules integrating L factor with the herpes simplex virus thymidine kinase gene.
- Transfection of these composite DNA molecules into various mouse cell lines.
- Analysis of plasmid establishment, copy number, structural integrity, and stability under prolonged culture conditions, with and without selective pressure.
Main Results:
- Composite DNA molecules were successfully established as plasmids in multiple mouse cell lines post-transfection.
- The established plasmids demonstrated structural stability during prolonged culture, maintaining a constant copy number even without selective pressure.
- A composite DNA lacking polyoma T antigen sequences was also established in a mouse L cell line expressing polyoma T antigen.
Conclusions:
- The L factor plasmid can be utilized as a stable vector for establishing foreign DNA in mammalian cells.
- The established composite plasmids exhibit remarkable stability and consistent copy number, independent of selective pressure.
- This suggests L factor-based vectors hold potential for applications in mammalian gene expression and biotechnology.