EGFP-containing vector system that facilitates stable and transient expression assays.

Current methods for the creation of cell lines that express a gene of interest generally involve co-selection for a resistance marker. This process usually requires 5–6 weeks to complete, during which time the culture must be maintained in selection media. Transient assays for gene expression often use cotransfection with another plasmid to identify transfected cells and/or to control for variation in transfection efficiency. We describe an expression system in which the gene of interest is cloned into a vector that contains a gene for enhanced green fluorescent protein (EGFP; CLONTECH Laboratories, Palo Alto, CA, USA). The system is convenient because expressing cells can be directly detected and/or separated within one day of the introduction of the genetic material. It is also flexible because it allows for transient or stable expression using transfection, electroporation or retroviralmediated gene transfer. We describe the use of this vector system to monitor the down-regulation of CD4 on T cells following expression of the nef gene of simian immunodeficiency virus (SIVmac239). We replaced the neomycin phosphotransferase gene of the retroviral vector pLXSN with the EGFP gene to create the vector pLXSG (Figure 1). Expression of EGFP in this vector is driven by the simian virus 40 (SV40) early promoter. To ensure high EGFP expression from this plasmid, the consensus eukaryotic translation initiation sequence (CGCCACCATGG) (4) was engineered immediately surrounding its start codon. An additional plasmid (pLnefSG, Figure 1) was created by inserting the nef gene of SIVmac239 into the multiple cloning site of pLXSG that is located immediately downstream of the Moloney murine leukemia virus (MMLV) 5′ long terminal repeat. pLXSG and pLnefSG were independently introduced into Jurkat T cells by electroporation. Exponentially growing cells were incubated with 50 μg of cesium chloride-purified plasmid in a 0.4cm gap cuvette and given a 200-V and 960-μF charge from a Gene Pulser (Bio-Rad, Hercules, CA, USA). By 5 h after electroporation, green fluorescent cells were apparent in the culture (data