A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells
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Abstract:
We describe a recombinant plasmid, pBBPY1, containing polyoma virus sequences which persists episomally in mouse hepatoma (MH) cells and can be shuttled between these cells and bacteria. This plasmid is composed of a subgenomic fragment of a polyoma virus mutant that includes two origins of replication; sequences of plasmid pML2; the xanthine-guanine phosphoribosyltransferase gene of Escherichia coli (Ecogpt) under the control of SV40 early-region promoter and RNA processing signals, providing a dominant selectable marker for mammalian transfection. MH cells from colonies growing in HAT medium (hypoxanthine, aminopterin and thymidine) were found to contain vector DNA molecules in an episomal state, the majority of them unrearranged. When HAT-selective pressure was applied for only 3 days, the resulting cells contained up to 50-100 copies of intact plasmid, i.e. 20-fold more than cells grown under standard selection conditions with continuous HAT-selective pressure. Contrary to standard conditions, transient selection does not alter the epithelial morphology nor ability of transfected hepatoma cells to produce albumin.
Insights
A novel plasmid, pBBPY1, enables efficient episomal persistence and high-copy amplification in mouse hepatoma cells using transient selection. This facilitates genetic manipulation and maintains cell function.
Area of Science:
- * Molecular Biology
- * Virology
- * Genetic Engineering
Background:
- * Development of episomal vectors for mammalian cell transfection is crucial for genetic studies.
- * Polyoma virus DNA elements offer unique replication and persistence properties.
- * Dominant selectable markers are essential for identifying successfully transfected cells.
Purpose of the Study:
- * To construct and characterize a novel episomal shuttle vector, pBBPY1, for use in mammalian cells.
- * To evaluate the efficiency of episomal DNA replication and persistence in mouse hepatoma cells.
- * To investigate the impact of transient selection on plasmid copy number and cell characteristics.
Main Methods:
- * Construction of the recombinant plasmid pBBPY1 incorporating polyoma virus and pML2 sequences.
- * Transfection of mouse hepatoma (MH) cells with pBBPY1 and selection using HAT medium.
- * Analysis of episomal DNA state, copy number, and cell morphology under different selection conditions.
Main Results:
- * The pBBPY1 plasmid was successfully constructed and demonstrated episomal persistence in MH cells.
- * Transient HAT selection (3 days) resulted in significantly higher plasmid copy numbers (50-100 copies) compared to continuous selection.
- * Transient selection preserved the epithelial morphology and albumin production capability of the transfected hepatoma cells.
Conclusions:
- * The pBBPY1 plasmid is an effective episomal vector for mouse hepatoma cells.
- * Transient selection is a superior method for achieving high plasmid copy numbers without compromising cell function.
- * This vector system offers a valuable tool for genetic manipulation in mammalian cells.