Transient overexpression of wild-type or mutant cell cycle regulatory factors is a quick and relatively easy means to derive important information regarding the function of the respective factor in regulating cell cycle checkpoint pathways. The general approach for these studies is to introduce an expression vector for the gene of interest into a relevant cell type and assess alterations in cell cycle progression. There are numerous means for introducing expression vectors into cells, including a number of different transfection methods as well as various viral vector technologies. Because it is impossible to detail the protocol for each of these different methods in this chapter, the use of the calcium phosphate method will be discussed as a prototype. The calcium phosphate method is relatively easy, it is efficient in a number of cell lines, and it is inexpensive.
