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Establishment of composite DNA derived from L factor as a plasmid in mouse embryonal carcinoma (F9) cells
K Nishimori1, T Kohda, J Fujiwara
1Institute of Applied Microbiology, University of Tokyo, Japan.
Abstract:
We have recently reported a mammalian cell plasmid (L factor) whose structure is related to that of polyomavirus (T. Kusano, H. Uehara, H. Saito, K. Segawa, and M. Oishi, Proc. Natl. Acad. Sci. USA 84:1789-1793, 1987). When composite DNA constructed from L factor and a foreign gene was introduced into mouse embryonal carcinoma (F9) cells by transfection, the DNA was reestablished in the cells as a plasmid. The reestablished plasmid DNA in F9 cells could be rescued in Escherichia coli. The plasmid-bearing cells underwent normal in vitro differentiation in response to retinoic acid. The efficiency of plasmid establishment of the L-factor-derived DNA and transcriptional and transient replicational activities were compared with those of similar composite DNA constructed from polyomavirus and an embryonal carcinoma mutant of polyomavirus which is permissive in F9 cells. The results suggest an inverse relationship between the efficiency of the plasmid establishment and the activity of gene expression controlled by the intrinsic enhancer-promoter of the DNA.
Insights
Researchers created a mammalian cell plasmid (L factor) that can be reestablished in mouse cells after transfection. This plasmid allows for normal cell differentiation and suggests an inverse relationship between plasmid establishment and gene expression activity.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- A mammalian cell plasmid, termed L factor, with structural similarities to polyomavirus has been previously identified.
- Understanding the behavior of extrachromosomal DNA elements in mammalian cells is crucial for gene therapy and synthetic biology.
Purpose of the Study:
- To investigate the establishment and behavior of a composite DNA plasmid (L factor and a foreign gene) in mouse embryonal carcinoma (F9) cells.
- To compare the efficiency of plasmid establishment and gene expression activities with DNA constructs derived from polyomavirus.
Main Methods:
- Transfection of mouse F9 cells with composite DNA (L factor-foreign gene).
- Rescue of reestablished plasmid DNA in Escherichia coli.
- Assessment of in vitro differentiation of plasmid-bearing cells in response to retinoic acid.
- Comparative analysis of plasmid establishment efficiency, transcriptional, and transient replication activities with polyomavirus-derived DNA.
Main Results:
- Composite DNA, when transfected into F9 cells, was reestablished as a plasmid and could be rescued in E. coli.
- Plasmid-bearing F9 cells exhibited normal in vitro differentiation.
- An inverse relationship was observed between the efficiency of plasmid establishment and the activity of gene expression driven by the DNA's intrinsic enhancer-promoter.
Conclusions:
- The L factor-derived plasmid can be stably established in mammalian cells and supports normal cellular functions like differentiation.
- The efficiency of plasmid establishment in F9 cells is inversely correlated with the strength of the enhancer-promoter elements within the DNA construct.