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.