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 have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold 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 expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.
Liu
X
X
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142, USA.
Constantinescu
S N
SN
Sun
Y
Y
Bogan
J S
JS
Hirsch
D
D
Weinberg
R A
RA
Lodish
H F
HF
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002469:Cell Separation; D003001:Cloning, Molecular; D017931:DNA Primers; D005434:Flow Cytometry; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008907:Mink; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D017931:DNA Primers; D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins