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Mosaic mice with teratocarcinoma-derived mutant cells deficient in hypoxanthine phosphoribosyltransferase
Abstract:
Mutagenized stem cells of a cultured mouse teratocarcinoma cell line were selected for resistance to the purine base analog 6-thioguanine. Cells of a resistant clone were completely deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), the same X-linked lesion as occurs in human Lesch-Nyhan disease. After microinjection into blastocysts of another genetic strain, the previously malignant cells successfully participated in normal embryogenesis and tumor-free, viable mosaic mice were obtained. Cells of tumor lineage were identified by strain markers in virtually all tissues of some individuals. Mature function of those cells was evident from their tissue-specific products (e.g., melanins, liver proteins). These mutagenized teratocarcinoma cells are therefore developmentally totipotent. Retention of the severe HPRT deficiency in the differentiated state was documented in extracts of mosaic tissues by depressed specific activity of the enzyme, and also by presence of unlabeled clones in autoradiographs of explanted cells incubated in [(3)H]hypoxanthine. Some mosaic individuals had mutant-strain cells in only one or a few tissues. Such animals may provide unique opportunities to identify the tissue sources of particular aspects of the complex disease syndrome. The tissue distribution of HPRT-deficient cells suggests that selection against them is particularly strong in blood of the mosaic mice, as is already known to be the case in human heterozygotes. This phenotypic parallelism supports the expectation that afflicted F(1) male mice that might be obtained from mutant germ cells can serve as a model of the human disease.
Insights
Mutagenized teratocarcinoma stem cells lacking hypoxanthine phosphoribosyltransferase (HPRT) enzyme activity were used to create mosaic mice. These mice demonstrate the developmental totipotency of these cells and offer a model for human Lesch-Nyhan disease.
Area of Science:
- Developmental biology
- Stem cell biology
- Genetics
Background:
- Teratocarcinoma stem cells offer a model for studying early development.
- Genetic mutations can lead to severe human diseases, such as Lesch-Nyhan disease, caused by HPRT deficiency.
- Understanding stem cell totipotency is crucial for regenerative medicine.
Purpose of the Study:
- To investigate the developmental totipotency of mutagenized mouse teratocarcinoma stem cells.
- To create a mouse model for human Lesch-Nyhan disease by inducing HPRT deficiency.
- To assess the potential of these cells in normal embryogenesis and tissue differentiation.
Main Methods:
- Teratocarcinoma stem cells were mutagenized and selected for resistance to 6-thioguanine, resulting in HPRT deficiency.
- Microinjection of these mutagenized cells into blastocysts of a different genetic strain.
- Analysis of mosaic mice for the presence and function of HPRT-deficient cells in various tissues.
- Enzyme activity assays and autoradiography to confirm HPRT deficiency in differentiated cells.
Main Results:
- Mosaic mice were successfully generated, with HPRT-deficient teratocarcinoma cells contributing to various tissues.
- These cells exhibited mature, tissue-specific functions, confirming their developmental totipotency.
- HPRT deficiency was retained in differentiated cells, and its distribution varied among mosaic individuals.
- Strong selection against HPRT-deficient cells was observed in the blood, mirroring human heterozygotes.
Conclusions:
- Mutagenized teratocarcinoma stem cells are developmentally totipotent and can integrate into normal embryogenesis.
- The generated mosaic mice serve as a valuable model for studying Lesch-Nyhan disease and the tissue-specific effects of HPRT deficiency.
- Further studies with these mosaic mice can elucidate the tissue origins of disease symptoms and explore therapeutic strategies.