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Multipotent mutator strain of mouse teratocarcinoma cells
Abstract:
A mutator strain [AraCr (1.5)4], isolated from mutagenized cultures of multipotent mouse teratocarcinoma cells (embryonal carcinoma stem cells), exhibited a dNTP pool imbalance, with more than a 10-fold relative increase in the intracellular concentration of dCTP. The increase in the spontaneous rate of mutation for 6-thioguanine resistance was 3.6-fold and for ouabain resistance, 7.9-fold. Normalization of the dCTP/dTTP ratio by addition of thymidine and deoxycytidine to the media was associated with normalization of the mutation rates. AraCr (1.5)4 cell retained its multipotency (including chimerization potential) when injected into blastocysts. Moreover, its differentiated progeny expressed the dNTP pool imbalance and mutator phenotype in vitro. The preliminary finding of an increased frequency of morphologically abnormal embryos derived from a series of transplanted blastocysts injected with AraC2 (1.5)4 stem cells is consistent with significant phenotypic effects in vivo.
Insights
A mouse stem cell strain with high dCTP levels showed increased mutation rates. Restoring dCTP balance normalized mutations, and the cells retained stem cell properties, indicating dNTP imbalance impacts mutation and development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Multipotent mouse teratocarcinoma cells (embryonal carcinoma stem cells) are crucial for developmental studies.
- dNTP pool imbalance is implicated in mutagenesis, but its role in stem cell function and development requires further investigation.
Purpose of the Study:
- To investigate the effects of dNTP pool imbalance on mutation rates and stem cell properties.
- To determine if dNTP pool imbalance affects developmental potential in vivo.
Main Methods:
- Isolation and characterization of a mutator mouse stem cell strain (AraCr (1.5)4) with elevated dCTP levels.
- Assessing mutation rates for drug resistance (6-thioguanine, ouabain).
- Evaluating stem cell multipotency through blastocyst injection and teratoma formation.
- Analyzing the effects of dNTP pool normalization via media supplementation.
Main Results:
- The AraCr (1.5)4 strain displayed a >10-fold increase in intracellular dCTP, correlating with 3.6-fold and 7.9-fold increases in mutation rates for 6-thioguanine and ouabain resistance, respectively.
- Normalization of the dCTP/dTTP ratio by adding thymidine and deoxycytidine restored normal mutation rates.
- AraCr (1.5)4 cells maintained multipotency and chimerization potential.
- Differentiated progeny of AraCr (1.5)4 cells exhibited the dNTP pool imbalance and mutator phenotype in vitro.
- Preliminary data suggest an increased frequency of morphologically abnormal embryos from blastocysts injected with AraCr (1.5)4 stem cells, indicating in vivo phenotypic effects.
Conclusions:
- A significant dNTP pool imbalance, specifically elevated dCTP, directly contributes to increased spontaneous mutation rates in mouse stem cells.
- The mutator phenotype and dNTP pool imbalance are stable traits that can be inherited by differentiated progeny.
- Despite the mutator phenotype, multipotency and developmental potential are retained, though potential in vivo developmental abnormalities warrant further investigation.