This work was undertaken to molecularly clone the Nirand strain of the polycythaemia inducing Friend spleen focus forming virus (F-SFFVp) together with its replication competent helper virus. Friend murine leukaemia virus (F-MuLV). To this aim viral extrachromosomal DNA molecules (of both linear and circular nature corresponding to both of these viruses) which could be induced in Friend cell lines were molecularly characterised with respect to their quantitative increase during differentiation and subcellular location. One cell line, F4-6 in which considerable amounts of these extrachromosomal DNAs could be detected was utilised for the large scale production and purification of both SFFVp and F-MuLV extrachromosomal DNA molecules. Restriction enzyme analysis of such molecules in combination with Southern blotting enabled the construction of primary restriction enzyme maps which allowed the selection of a molecular cloning strategy. Both of these viruses were subsequently molecularly cloned and were subsequently shown to be biologically active after transfection into recipient cells. A further aspect of this work was to construct a biologically active SFFVp containing a dominant selectable marker gene. A selectable construct was generated by joining different regions of the genome of the myeloproliferative sarcoma virus (MPSV) and SFFVp. A construct with the U3 region from the long terminal repeat (LTR) of SFFVp and the envelope gene region (gp55) of SFFVp (designated neo2 SFFVp) was found to be fully active as a selectable retroviral vector with identical biological properties to the wild type SFFVp neo2 SFFVp induced erythroid differentiation in vivo, with infected cells no longer requiring erythropoietin for differentiation. Furthermore neo2 SFFVp infected spleen cells could be used to generate immortal Friend leukaemia cells which were selectable with geneticin (neo2). A further construct, neo2 SFFV-M, which had a U3 region originating from NPSV was able to cause erythropoietin independent erythroid differentiation. However as compared to neo2 SFFVp and indeed the wild type SFFVp, neo2 SFFV-M was able to induce the erythroproliferative disease with a different kinetics. All attempts to isolate transformed Friend cells with this construct failed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:290376 |
| Date | January 1989 |
| Creators | Hunt, Nicholas |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/108015/ |
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