Transgenic sheep 229 carries one copy of a mouse metallothionein-I promoter HSV-tk fusion gene (pMK), Poly A<SUP>+</SUP> RNA from a liver biopsy revealed no HSV-tk message. A skin fibroblast cell-line was established from sheep 229 and is the subject of this thesis. The cell-line is non-immortal and senesces after 30-40 population doublings <i>in vitro</i>. The cell-line carries one intact copy of pMK which is not rearranged as detected by southern blot analysis of genomic DNA. The cells do not display any HSV-tk activity and no HSV-tk mRNA is detected by northern analysis. The expression of the sheep metallothionein gene family in the cell-line was investigated using gene specific probes. All four known active members are expressed and induced in response to zinc. The two major transcripts, from the Ia and II genes, are also induced by copper. The basal level and zinc induced expression of the gene family is not significantly altered by transformation of the cells by transfecting plasmids encoding the SV40 early region. When mouse metallothionein-I promoter fusion genes (including pMK) are introduced into the cell-line (by electroporation) both expression and zinc regulation is noticed. Based upon stable transformation efficiency the mouse metallothionein-I promoter is 3-4 times less efficient than the SV40 early region promoter. Immuno-staining was used to detect the transient expression of the SV40 large T antigen (Tag) driven by either the SV40 early region (pSV3gpt) or the mouse metallothionein promoter (pMTLT). In the absence of zinc the number of cells detectably expressing pMTLT is 2% of that expressing pSV3gpt, following zinc induction the number of cells expressing pMTLT increases to 20% of that expressing pSV3gpt. The mMT-I promoter is therefore deficient in the establishment of expression compared to the SV40 early region promoter in sheep cells. Since both the endogenous sheep metallothionein genes and mouse metallothionein-I promoter fusion genes are active in the cell-line, itis concluded that an overriding, negative position effect is the most likely explanation for the failure of the pMK transgene to be expressed. The ability of viral and cellular oncogenes to transform the cell-line was investigated. Expression of SV40 Tag causes the cells to divide more rapidly, loose contact inhibition and anchorage dependent growth but does not lead to immortalisation. Clones established which express SV40 Tag have a vastly increased proliferative capacity. However, after 60-70 population doublings all clones enter crisis and die. Failure to express SV40 Tag is not the cause of crisis. No stable phenotypic effect is noticed when activated Ha-ras and c-myc (singly or in conjunction) or the bovine papilloma virus transforming region are introduced. A transforming murine p53 clone severely inhibits the plating efficiency of the cells. Attempts were made to re-claim the transgene for further analysis. Screening of a random genomic library failed to detect any positive clones. Attempts to clone a Hind III fragment containing the whole transgene from size fractionated DNA enriched for the fragment, were also unsuccessful.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:662094 |
| Date | January 1990 |
| Creators | Smith, Paul David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12989 |
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