Design and in vitro testing of macrophage-specific retroviral vectors.

(A) Schematic representation of RVs used in this study. All RVs contain LTRs and packaging signals (ψ⁺) derived from the Moloney murine leukemia virus. PBM-I-EGFP is a standard, non–cell type–specific RV in which expression is driven by the viral 5′ LTR.23 This vector contains an internal ribosomal entry site (IRES) in addition to the cDNA encoding EGFP. The other RVs have a deletion of essential promoter and enhancer elements (indicated by gray shading) in the 3′ LTR, making them self-inactivating (SIN). In these vectors, gene expression is solely driven by a promoter containing either 2.9 kb (CD68L) or 342 bp (CD68S) of sequence 5′ to the ATG initiation codon of the CD68 gene, in addition to the CD68 first intron (IVS-1). The direction of transcription, indicated by the bent arrow, is opposite that of the viral 5′ LTR. In addition to cDNAs encoding either an HA epitope–tagged version of EGFP or ApoE, the RVs contain a bovine growth hormone polyadenylation signal (pA) to terminate RNA transcription. All of the RVs are in plasmids that contain expression cassettes for the puromycin resistance gene and the Epstein-Barr virus nuclear antigen (EBNA) in addition to the EBNA origin of replication (not shown). This allows selection of packaging cell lines with episomal copies of the plasmid.25 (B) Bone marrow–derived mφs (BMDMs) were transduced with each of the different retroviral constructs, and EGFP expression was analyzed by fluorescence-activated cell sorter (FACS). The percentage of EGFP⁺ cells was determined using a gate set to exclude nontransduced control cells. Data are from a single experiment performed in duplicate and are representative of 3 similar experiments.