Stable expression of fluorescently tagged proteins for studies of mitosis in mammalian cells

摘要

Cultured mammalian cells have been a popular and useful system for studying mitosis(1-3). In particular, the direct observations of fluorescently tagged cytoskeletal and chromosomal proteins in live cells have furthered our understanding of the dynamics of cell division(4-6). Although cultured cells are not suitable for manipulation using genetic methods, they are simply grown and maintained in the laboratory and easily transfected with dominant-negative or constitutively active constructs, and RNA interference can be used to selectively reduce or eliminate the expression of specific genes. Cultured cells are somatic rather than embryonic and have an extended G1 phase of the cell cycle. They are therefore ideal for studies of the regulation of the somatic cell cycle (Figs. 1 and 2). This protocol describes methods for generating cell Lines that constitutively express green fluorescent protein (GFP)-tagged proteins. Although transient transfection is relatively fast, the expression level of the GFP fusion protein under study may vary greatly from cell to cell. Establishing permanent cell tines has the advantage that at cells express the chimeric protein at the same level and each cell line can be characterized. Permanent cell Lines are especially useful for observation of cellular events that could be altered by overexpression of a GFP fusion protein. Finally, a major challenge for those who study mitosis is the need to keep cells alive and actively mitotic during observations. The protocol therefore also incorporates methods for maintaining healthy, dividing cells in culture and during imaging.