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Updated: Jul 28, 2026
Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Published on: February 24, 2014
Use of microsatellite DNA polymorphisms on mouse chromosome 11 for in vitro analysis of thymidine kinase gene
M C Liechty1, Z Hassanpour, J C Hozier
1Applied Genetics Laboratories Inc., Melbourne, FL 32901.
Abstract:
The mouse lymphoma (L5178Y tk+/- 3.7.2C) in vitro mutagenesis assay can measure the genotoxic effects of a wide variety of chemical agents by inactivation of a single functional thymidine kinase (tk-1) gene. We have previously demonstrated, using cytogenetic and molecular techniques, that the types of molecular lesions associated with tk-1 gene inactivation span a wide range similar to that seen in tumor cells at specific oncogene and tumor suppressor gene loci. We have identified, using polymerase chain reaction techniques, 21 microsatellite, or 'simple sequence repeat', polymorphisms between chromosomes 11a and 11b in 3.7.2C cells. These microsatellite polymorphisms span virtually the entire chromosome, from mapping positions of 3-78 centiMorgans (cM) from the centromere, thus providing landmarks to study loss of genetic material across the entire chromosome. Four of the microsatellite polymorphisms lie within 12 cM of tk-1, and provide a means of mapping loss of genetic material in the immediate vicinity of tk-1, a capability that we have not previously had in the mouse lymphoma assay. Loss of alleles (i.e. loss of heterozygosity) is an important feature of tumor development, having to do with tumor suppressor gene expression. Therefore, the ability to detect loss of heterozygosity in the mouse lymphoma assay will make the assay an extremely valuable tool in the detection of agents capable of inducing loss of heterozygosity.
Insights
The mouse lymphoma assay detects genotoxic chemical effects by measuring thymidine kinase (tk-1) gene inactivation. New microsatellite markers enable mapping of genetic material loss near tk-1, enhancing detection of agents causing loss of heterozygosity.
Area of Science:
- Toxicology
- Genetics
- Molecular Biology
Background:
- The mouse lymphoma assay (L5178Y tk+/- 3.7.2C) assesses genotoxicity via thymidine kinase (tk-1) gene inactivation.
- Previous studies showed molecular lesions in tk-1 inactivation resemble those in tumor suppressor genes.
- Cytogenetic and molecular techniques have been used to study these lesions.
Purpose of the Study:
- To develop new tools for the mouse lymphoma assay to study genetic alterations.
- To identify microsatellite polymorphisms on mouse chromosomes 11a and 11b.
- To map loss of genetic material in the vicinity of the tk-1 gene.
Main Methods:
- Utilized polymerase chain reaction (PCR) techniques to identify microsatellite polymorphisms.
- Mapped 21 microsatellite polymorphisms across mouse chromosomes 11a and 11b.
- Focused on polymorphisms within 12 centimorgans (cM) of the tk-1 gene.
Main Results:
- Identified 21 microsatellite polymorphisms spanning chromosomes 11a and 11b.
- Located four polymorphisms within 12 cM of the tk-1 gene, providing proximity mapping.
- Established landmarks for studying loss of genetic material across the chromosome.
Conclusions:
- The identified microsatellite polymorphisms enhance the mouse lymphoma assay's capability.
- This advancement allows for precise mapping of genetic material loss near tk-1.
- The improved assay is valuable for detecting agents that induce loss of heterozygosity, crucial in tumor development.
