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Title: Correction of ERCC1 deficiency in mice
Author: Selfridge, Jim
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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A novel ERCC1 mRNA has been identified in mouse skin. Subsequent characterisation of the transcript demonstrated that the difference between the normal and skin-specific ERCC1 mRNA was at the 5' end and is due to differential initiation of transcription. As with the normal ERCC1 transcript the novel skin-specific transcript did not appear to be induced by UV irradiation. A functional ERCC1 minigene was constructed to facilitate subsequent analysis of the upstream promoter region and identification of the sequences involved in the regulation of the observed skin-specific pattern of expression. The minigene corrected the UV sensitivity of ERCC1-deficient cultured cells but did not exhibit the characteristic skin-specific expression pattern at the level of mRNA analysis. A pilot study for a potential gene therapy approach to increasing the lifespan of the ERCC1 knockout mice was completed. Recombinant ecotropic retroviruses containing ERCC1 coding sequences were produced using a murine leukaemia virus derived viral vector and viral packaging cell line. The resultant ERCC1 retrovirus was shown to partially correct the UV sensitivity associated with pools of ERCC1-deficient mouse embryonic fibroblasts. Clones, subsequently isolated from the transduced pools, were shown to be phenotypically corrected to wild type levels. The observed phenotypic correction correlated with expression of a retroviral ERCC1 mRNA. A transthyretin regulated ERCC1 transgene was used to bring about the targeted expression of ERCC1 in the liver. Pronuclear injection of the- transgene was used to produce a transgenic mouse line containing ~5 copies of the transgene integrated at a single site. Expression of this transgene on an ERCC1 -deficient background resulted in the correction of the lethal liver phenotype. Transgene positive nulls were not as severely runted and survived for between 9 and 12 weeks compared to the three-week survival of the transgene negative ERCC1-deficient mice. The consequences of ERCC1 deficiency in other tissues were studied. Abnormalities were identified in the skin and kidneys of adult transgene positive nulls. An investigation into the consequences of DNA repair deficiency on UV-B induced immunosuppression revealed that antigen presenting epidermal Langerhans cells, in the transgene positive nulls, did not show the normal pattern of accumulation in the lymph nodes following UV irradiation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available