A mouse model system was developed to sensitively detect mutagenic events arising spontaneously in mouse fibrosarcoma cells grown under in vitro or in vivo conditions. These cells were genetically manipulated to enhance their sensitivity as detectors of mutational events at the X-linked hprt (hypoxantine phosphoribosyltransferase) gene, a frequently used marker of genomic instability. A fibrosarcoma cell with three X-chromosomes and randomly distributed X-specific fragments was isolated from a male mouse. Initially, this cell line was resistant to mutation induction, presumably due to the presence of multiple hprt genes. Inactivation of all but one hprt gene resulted in a cell line with a heterozygous marker which was at least 1000 times more sensitive to detecting induced mutational events. Infection of this derived cell line with a selectable marker for neomycin resistance allowed recovery of cells which subsequently demonstrated that the tumour environment was capable of enhancing spontaneous in vitro mutation induction. A human lymphoblastoid cell line was also genetically manipulated to establish nine stable transfectants as models for the study of genomic instability. The endogenous X-linked hprt gene was mutated, presumably by a large deletion, and subsequently transfected with a human hprt minigene encoding for its own promoter and poly-adenosine signal. Transfectants were assessed for the stability of the transgene. Transfected clones demonstrated a 2 to 600 fold increase in spontaneous mutation rate at the transgene loci compared to the endogenous hprt locus. There was no evidence of mutator phenotype activation in any of the clones as judged by testing at another locus, the endogenous tk (thymidine kinase) gene. An inverse correlation was also noted between the number of the inserted sequences and the stability of gene expression, with single gene insertions being most stable and tandem insertions being the least stable.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/4281 |
| Date | January 1997 |
| Creators | Wilkinson, Diana. |
| Contributors | Birnboim, C., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 194 p. |
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