A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells

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.