Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency.

Area of Science:

• Molecular Biology
• Genetics
• Toxicology

Background:

• Assessing mutagen-induced DNA damage in human cells is crucial for understanding genetic toxicology.
• Existing methods for analyzing mutations can be complex and time-consuming.

Purpose of the Study:

• To develop a novel recombinant DNA shuttle vector for the molecular analysis of induced mutations in human cells.
• To establish a system for quantifying mutation frequencies and responses to mutagens.

Main Methods:

• A recombinant DNA shuttle vector containing the herpes simplex virus type 1 thymidine kinase (HSV tk) gene was constructed.
• The vector was introduced into Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL-721) via electroporation.
• Mutation frequencies in the HSV tk gene were analyzed after exposure to N-ethyl-N-nitrosourea.

Main Results:

• The shuttle vector replicated in both Escherichia coli and human lymphoblastoid cell lines.
• A high percentage of transfected cells expressed the vector-encoded hygromycin resistance gene.
• A baseline mutation frequency of 6 X 10(-5) was observed in the HSV tk gene.
• Treatment with N-ethyl-N-nitrosourea induced a dose-dependent, up to 15-fold increase in mutation frequency, paralleling cellular gene mutation responses.

Conclusions:

• The developed shuttle vector is effective for molecular analysis of induced mutations in human cells.
• This system provides a sensitive and quantitative method for assessing mutagenicity.
• The findings support the utility of this vector in genetic toxicology and cancer research.