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Molecular analysis of deletion mutations at the HPRT locus in CHO cells using the polymerase chain reaction
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Darroudi, F., D.K. Benova and A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlands) A novel culture system for evaluating chromosomal alterations in mouse spleen cells
In developing models for human genetic toxicology studies, it is important to determine the persistence of the damage in the target cells in an animal model. In this study we present a novel method to culture mouse spleen cells. The spleen cells were stimulated to divide using a combined treatment of 2 mitogens, concanavalin A and purified phytohemagglutinin (PHA 16), that lead to improved metaphase yield. Spleen cells generally are representative of peripheral blood lymphocytes, they are relatively long-lived and initially are non-dividing. Experiments were conducted to study the effect of Xirradiation on mouse spleen cells with respect to the induction of chromosomal aberrations and micr0nuclei (MN). Dose-response curves were obtained following irradiation of G1 or G 2 cells in vitro. In addition mice were whole-body X-irradiated in vivo with the same doses, so that an in vitro-in vivo comparison could be made. The results indicate that mouse spleen cells irradiated either in vivo or in vitro responded similarly to X-irradiation as measured by dicentric formation in C-banded chromosomes. In addition, the frequency of MN was determined in cytochalasin B-mediated binucleated cells following X-ray treatment of spleen cells in vitro. This culture system proved to be reliable and reproducible, and generally yielded adequate numbers of mitotic cells for analyzing chromosomal alterations following in vitro or in vivo exposure to chemical or radiation.
versity of Texas Medical Branch, Galveston, 2 Institute of Molecular Genetics, Baylor College of Medicine, Houston and 3 Laboratories for Genetic Services, Inc., Houston, TX (U.S.A.) Molecular analysis of deletion mutations at the HPRT locus in CHO cells using the polymerase chain reaction We have developed a rapid screening method using triplex DNA amplification via the polymerase chain reaction (PCR) for the detection of deletion mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus in CHO cells. Three sets of primers were used to simultaneously amplify DNA fragments flanking exon 3 and exon 9 of the hprt gene, and a portion of the adenine phosphoribosyltransferase (aprt) gene which served as an internal positive control. PCR products from 120 mutants were analyzed by electrophoresis on agarose gels for the presence or absence of the corresponding DNA fragments. Of the 82 spontaneous mutants, 75 did not exhibit distinguishable differences from the parental CHO-K1-BH4 cells, 4 were found to have deletions at exon 9, and 3 showed deletions in both exon 3 and exon 9. Among the 38 X-ray-induced mutants, 18 showed normal amplification patterns, and 20 had deletions. Of these, 12 mutants showed deletions at both exons, and another 8 mutants had deletions only at exon 9. Deletions occurred most frequently at the 3' end (exon 9) of the hprt gene in the X-ray-induced mutants, suggesting an existence of hot spots for deletions in this region. This technique provides a simple method for the rapid discrimination of deletion mutations from point mutations at the hprt locus, which is particularly useful for the detection of deletions in mutants induced by ionizing radiation and other radiomimetic chemicals.
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Xu, Z.D. 1.3, y . Yu 1,3, B.J.F. Rossiter 2, R.A. Gibbs 2, C.T. Caskey 2 and A.W. Hsie a, 1 Uni-
Lichtenauer-Kaligis, E.G.R., P van de Putte, M. Giphart-Gassler and J.G. Tasseron-de Jong, Laboratory of Molecular Genetics, Leiden University, Leiden (The Netherlands)
Darroudi, F., D.K. Benova and A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlands) A novel culture system for evaluating chromosomal alterations in mouse spleen cells
In developing models for human genetic toxicology studies, it is important to determine the persistence of the damage in the target cells in an animal model. In this study we present a novel method to culture mouse spleen cells. The spleen cells were stimulated to divide using a combined treatment of 2 mitogens, concanavalin A and purified phytohemagglutinin (PHA 16), that lead to improved metaphase yield. Spleen cells generally are representative of peripheral blood lymphocytes, they are relatively long-lived and initially are non-dividing. Experiments were conducted to study the effect of Xirradiation on mouse spleen cells with respect to the induction of chromosomal aberrations and micr0nuclei (MN). Dose-response curves were obtained following irradiation of G1 or G 2 cells in vitro. In addition mice were whole-body X-irradiated in vivo with the same doses, so that an in vitro-in vivo comparison could be made. The results indicate that mouse spleen cells irradiated either in vivo or in vitro responded similarly to X-irradiation as measured by dicentric formation in C-banded chromosomes. In addition, the frequency of MN was determined in cytochalasin B-mediated binucleated cells following X-ray treatment of spleen cells in vitro. This culture system proved to be reliable and reproducible, and generally yielded adequate numbers of mitotic cells for analyzing chromosomal alterations following in vitro or in vivo exposure to chemical or radiation.
versity of Texas Medical Branch, Galveston, 2 Institute of Molecular Genetics, Baylor College of Medicine, Houston and 3 Laboratories for Genetic Services, Inc., Houston, TX (U.S.A.) Molecular analysis of deletion mutations at the HPRT locus in CHO cells using the polymerase chain reaction We have developed a rapid screening method using triplex DNA amplification via the polymerase chain reaction (PCR) for the detection of deletion mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus in CHO cells. Three sets of primers were used to simultaneously amplify DNA fragments flanking exon 3 and exon 9 of the hprt gene, and a portion of the adenine phosphoribosyltransferase (aprt) gene which served as an internal positive control. PCR products from 120 mutants were analyzed by electrophoresis on agarose gels for the presence or absence of the corresponding DNA fragments. Of the 82 spontaneous mutants, 75 did not exhibit distinguishable differences from the parental CHO-K1-BH4 cells, 4 were found to have deletions at exon 9, and 3 showed deletions in both exon 3 and exon 9. Among the 38 X-ray-induced mutants, 18 showed normal amplification patterns, and 20 had deletions. Of these, 12 mutants showed deletions at both exons, and another 8 mutants had deletions only at exon 9. Deletions occurred most frequently at the 3' end (exon 9) of the hprt gene in the X-ray-induced mutants, suggesting an existence of hot spots for deletions in this region. This technique provides a simple method for the rapid discrimination of deletion mutations from point mutations at the hprt locus, which is particularly useful for the detection of deletions in mutants induced by ionizing radiation and other radiomimetic chemicals.
87 86
Xu, Z.D. 1.3, y . Yu 1,3, B.J.F. Rossiter 2, R.A. Gibbs 2, C.T. Caskey 2 and A.W. Hsie a, 1 Uni-
Lichtenauer-Kaligis, E.G.R., P van de Putte, M. Giphart-Gassler and J.G. Tasseron-de Jong, Laboratory of Molecular Genetics, Leiden University, Leiden (The Netherlands)