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Title: Use of gene targeting to study the mouse ERCC1 gene
Author: Hsia, Kan-Tai
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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The main aims of this work to set up two new mouse models, by conventional and conditional gene targeting, to investigate the functions of ERCC1, which has not, thus far, been associated with any known human disorders. To facilitate gene targeting studies, a series of overlapping ERCC1 genomic clones were retrieved from a bacteriophage library. Analysis of the mouse ERCC1 5'-flanking region revealed a novel transcription initiation site. Furthermore, several potential liver- and brain-specific transcription factor binding sites were present. A novel ERCC1-deficient mouse line, designated ET#209, was generated by the targeted deletion of the ERCC1 exon 3-5 region. In addition to the aberrant nuclei in the KT#209 ERCC1-/- livers, as reported in previous ERCC1 knockouts, we also found microvesicular steatosis and mitochondrial abnormalities in livers of both our ERCC1-deficient lines. These findings suggested that oxidative stress may damage nuclear and/or mitochondrial DNA, leading to the abnormal lipid deposits. We also found that other repair proteins (MSH2, PMS2 and PCNA) and an immediate early gene product, c-fos, were elevated in both ERCC1-deficient liver extracts. A delay in cerebellar development was found in both our ERCC1-deficient lines and the possibility of a cerebellar disorder was investigated. Also, we have demonstrated that some thymocyte markers were changes in flow cytometric profiles, suggesting a possible role for ECC1 in V(D)J recombination. To overcome the limitations of premature death in the ERCC1-deficient mice, we used a conditional gene targeting strategy. We reconstructed a functional ERCC1 allele with loxP sites flanking exons 3 and 5. In vitro analysis demonstrated that the ERCC1 exon 3-5 region between the loxP sites was deleted after transient expression of Cre recombinase. We have generated mice with this floxed ERCC1 allele, which will be used for Cre/loxP-mediated recombination in a tissue- and/or developmentally-specific manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available