Related Experiment Videos
Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences
Abstract:
To study the mutator phenotype characteristic of tumors showing widespread replication errors at simple DNA repeat sequences (RER+), we designed a selectable reporter system for the detection of such mutations in mammalian cells. A hygromycin B phosphotransferase gene was rendered out-of-frame by the insertion of a (CA)13 dinucleotide repeat tract immediately following the ATG start codon, and subcloned into a retroviral expression vector containing a G418 (neo) selectable marker. Following transduction of this construct into cultured cells, clonal neo+ cell lines were established and then tested for their ability to form colonies in hygromycin B-containing medium. Using this system, we found that the HCT116, LS174T and LS180 human colon carcinoma cell lines acquire hygromycin resistance (hygr) at a 100-fold higher frequency than the HT29, SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblast cell lines, and at a 25-fold higher rate than the Rat 6 embyro fibroblast cell line. DNA sequence analysis indicated that frameshift mutations had occurred within the CA dinucleotide repeat tract in HCT116 cells that became hygr. Thus, the mutation rates at simple repeated sequences in mammalian cell lines can be readily determined and studied using this system.
Insights
Researchers developed a reporter system to detect replication errors in DNA. This system identified specific human colon cancer cell lines with a high mutation rate at simple DNA repeats, aiding the study of the mutator phenotype.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Tumors with widespread replication errors at simple DNA repeat sequences exhibit a mutator phenotype.
- Detecting and quantifying these errors in mammalian cells is crucial for understanding cancer development.
Purpose of the Study:
- To design and validate a selectable reporter system for detecting mutations in simple DNA repeat sequences in mammalian cells.
- To assess the mutation rates in various human colon carcinoma and fibroblast cell lines.
Main Methods:
- A retroviral expression vector was engineered with a hygromycin B phosphotransferase gene rendered out-of-frame by a (CA)13 dinucleotide repeat tract.
- The construct included a G418 (neo) selectable marker for initial cell line establishment.
- Transduced cell lines were selected for G418 resistance and then tested for hygromycin B resistance to quantify mutation frequency.
Main Results:
- The HCT116, LS174T, and LS180 human colon carcinoma cell lines showed a 100-fold higher frequency of acquiring hygromycin resistance compared to other tested cell lines.
- DNA sequence analysis confirmed frameshift mutations within the CA dinucleotide repeat tract in resistant HCT116 cells.
- The developed system effectively determined mutation rates at simple repeated sequences in mammalian cell lines.
Conclusions:
- The reporter system is effective for studying the mutator phenotype associated with replication errors at simple DNA repeats.
- Specific human colon cancer cell lines demonstrate significantly elevated mutation rates at these sequences.
- This system provides a valuable tool for investigating DNA replication fidelity and its role in tumorigenesis.