Investigation into the role of c-MYC in differentiation of mouse embryonic stem cells

Abstract

The protein products of the myc family of proto-oncogenes regulate a wide variety of cellular processes, such as proliferation, apoptosis and cell growth. The importance of this family of genes is highlighted by the observation that their absence in mouse development results in significant defects and death during early embryogenesis. Investigation into the events of early embryogenesis can be modelled in the laboratory via the use of mouse embryonic stem (mES) cells, which are derived from the inner cell mass (ICM) of the mouse embryo and are equivalent to 4.5 days post coitum (dpc). An important characteristic of ES cells is that they are pluripotent, meaning that they have the potential to form every embryonic cell type. Maintenance of a pluripotent state, in culture is achieved by the exposure to the cytokine, Leukemia Inhibitory Factor (LIF). Differentiation of ES cells can achieved in cell culture in various ways, one of which leads to the formation of structures called embryoid bodies. These can be used as an in vitro model for embryonic differentiation of cells of the pluripotent ICM to the three primary germ layers, Ectoderm, Endoderm and Mesoderm. In the embryo and embryoid bodies it has be demonstrated that the differentiation of pluripotent cells into the germ layers there is a deceleration in the cell cycle times and adoption of a more tightly regulated cell cycle. It was the focus of this research to investigate the role of c-Myc protein in the differentiation of pluripotent embryonic stem cells into the three primary germ layers. To analyse the association of c-Myc protein with changes in cell cycle kinetics Western Blot Analysis was utilised. In this experiment, two distinct protein species were detected in early time points but were down-regulated as differentiation proceeded. Based on the estimated size of the proteins detected it was proposed that one of the bands was fulllength c-Myc and the other was the shorter, differentially translated c-Myc subtype, cMycS. Specific antibodies generated in the course of this research demonstrated that the shorter band detected in the Western Analysis was c-MycS, which illustrated that translation of the c-Myc protein is differentially regulated during differentiation of ES cells. 1bis thesis also describes the impact of c-Myc over-expression on differentiation and the maintenance of pluripotency. This effect was analysed during embryoid body differentiation experiments utilising No1ihern blot detection of RNA markers, which showed delayed differentiation kinetics. LIF titration assays demonstrated that c-Myc overexpression decreased dependence on LIF for ES cells to maintain pluripotency.