High spontaneous mutation frequency in shuttle vector sequences recovered from mammalian cellular DNA

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The shuttle vector pSV2gpt is a tool for gene transfer and expression studies.
• Stable transformants are crucial for investigating gene function and genetic alterations.
• Heterokaryon fusion allows for the study of genetic material in a mixed cellular environment.

Purpose of the Study:

• To investigate the stability and mutational frequency of the recombinant shuttle vector pSV2gpt in mammalian cells.
• To analyze the integrity of the Escherichia coli gpt gene within the plasmid after propagation in V79 and COS-1 cells.
• To determine the impact of cell fusion on plasmid DNA integrity and mutation rates.

Main Methods:

• Introduction of pSV2gpt into V79 Chinese hamster cells and selection of stable transformants.
• Identification and fusion of transformants with tandem duplications to simian COS-1 cells.
• Recovery of plasmid DNA from heterokaryons and transformation of E. coli HB101 (Gpt- derivative).
• Determination of the frequency of plasmids with mutations in the gpt gene.

Main Results:

• Stable V79 transformants expressing the E. coli gpt gene were successfully generated.
• Two transformants with tandem duplications of the plasmid were identified and utilized for cell fusion.
• Plasmid DNA recovered from heterokaryons showed a high frequency (approximately 1%) of mutations in the gpt gene.
• Similar mutation frequencies were observed when plasmid was recovered from COS-1 cells transfected with pSV2gpt.
• The majority of mutant plasmids exhibited rearrangements within the gpt gene region.

Conclusions:

• The pSV2gpt shuttle vector, after propagation in mammalian cells (V79 and COS-1), is prone to mutations and rearrangements, particularly within the gpt gene.
• Cell fusion (heterokaryon formation) does not significantly alter the observed mutation frequency of the plasmid compared to direct transfection.
• These findings highlight the potential instability of shuttle vectors in mammalian systems and the utility of the gpt gene as a reporter for genetic alterations.