A study on exogenous gene transfer in mammals

This thesis describes the development and utilisation of a transgenic system, a technology that was not established at Glasgow Veterinary School prior to the commencement of these studies. Satisfactory embryo culture is essential for the production of transgenic mice and Chapter 2 describes this aspect of the work. In this chapter different media were compared and protocols for culturing mouse embryos were established. Various aspects of the system were tested to ensure that conditions were optimal for embryo culture. Chapter 3 describes the generation of transgenic mice using pronuclear microinjection and those aspects of the system that influence the efficacy of this procedure were examined. Pronuclear microinjection is associated with immediate and delayed mortality of the embryos, this chapter concentrates on the timing of the embryo loss and discusses the causes of post-injection embryo and foetal death. The experience gained from the work described in Chapter 2 and 3 was adapted to investigate transient transgene expression in the murine preimplantation embryos using the bacterial lac Z reporter gene, the results of which are presented in Chapter 4. As preimplantation development proceeded there was an observed decline in the activity of the lac Z product -- beta-galactosidase. The chapter concludes with an investigation into the effects of DNA methylation on the expression of the introduced construct. Chapter 5 describes an experiment designed to investigate the potential of retroviruses to act as vectors for exogenous gene transfer in the ovine species. Wild type Feline Leukaemia Virus (FeLV) was used to infect early ovine embryos and subsequent analysis of foetuses was used to assess the efficacy of introducing viral gene sequences into the mammalian genome. Two out of the 17 foetuses examined contained FeLV proviral sequences. The transgenic system was utilised to investigate the role of the human myc proto-oncogene in the induction of murine T-cell leukaemia, this work is presented in Chapter 6. This is the first study to exclusively target murine T-cells with the c-myc oncogene and these transgenic mice developed thymic lymphoma at low frequency despite the apparent absence of transgene expression in healthy pre-lymphomatous mice. However the presence of the oncogene accelerates Murine Leukaemia Virus (MuLV) induced lymphoma development. The results presented in Chapter 6 indicate that the exogenous myc oncogene can co-operate in the tumourigenic process but that subsequent events are required for the development of the fully malignant phenotype.