Generation of mammalian cells stably expressing multiple genes at predetermined levels

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression h ave been difficult to achieve, especially for cell lines with low transfect ion efficiency or when expression of multiple genes is required. An interna l ribosomal entry site (IRES) has been incorporated into many types of expr ession vectors to allow simultaneous expression of two genes. However, ther e has been no systematic quantitative analysis of expression levels in indi vidual cells of genes linked by an IRES, and thus the broad use of these ve ctors in functional analysis has been limited. We constructed a set of retr oviral expression vectors containing an IRES followed by a quantitative sel ectable marker such as green fluorescent protein (GFP) or truncated cell su rface proteins CD2 or CD4. The gene of interest is placed in a multiple clo ning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the similar to 100-fo ld differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of exp ression of the gene downstream of the IRES and the expression level and fun ctional activity of the gene cloned upstream of the IRES are highly correla ted in stably infected target cells. This feature makes our vectors extreme ly useful for the rapid generation of stably transfected cell populations o r clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of express ion of the gene downstream of the IRES. We show how these vectors can be us ed to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressin g two different cloned proteins, Ski and Smad4. Correlation of a biologic e ffect with the level of expression of the protein downstream of the IRES pr ovides strong evidence for the function of the protein placed upstream of t he IRES. (C) 2000 Academic Press.