Chapter 20 Nuclear Transplantation with Mammalian Cells

Publisher Summary This chapter describes the techniques required to construct large, viable, homogeneous populations of true cytoplasmic-nuclear hybrids. These techniques are being applied to the construction of novel systems for investigating gene expression and its control in mammalian cells. Thus, the cytoplasts and karyoplasts prepared from various differentiated and transformed cell lines that exhibit distinct morphological and/or biochemical traits are fused to form true cytoplasmic-nuclear hybrid cells. The hybrids and their progeny are analyzed for the maintenance, loss, or acquisition of the parental cell traits. The chapter outlines the methods for the Preparation and Characterization of Karyoplasts and of Cytoplasts. The procedure for nuclear transplantation involves the preparation of Sendai virus, virus-mediated fusion, characterization of hybrids, and genetic selection techniques. The selection procedure should be applicable for the selection of cytoplasmic-nuclear hybrids. Thus, before fusing cytoplasts and karyoplasts derived from cell lines of interest, the mutants resistant to appropriate drugs, and toxins can first be selected.