Figure 2.
AAV vectors used in this study. Components of the vectors have been described previously.6,21 The inducible Epo reporter vector AAV-Z12I-rhEpo-3 (A) consists of 12 binding sites for the ZFHD1 DNA binding domain, a minimal interleukin-2 promoter (Min. IL2), a chimeric intron, the rhesus Epo (rhEpo) coding sequence, and an SV40 late gene 3′ untranslated region (UTR). The transcription factor construct AAV-CMV-TF1 (B) contains a human cytomegalovirus (CMV) enhancer driving expression of a bicistronic gene with the following components: an activation domain fusion (FRAP*, the FRB fragment of human FRAP [mTOR] in which threonine 2098 is mutated to leucine, fused to an activation domain derived from the p65 subunit of human nuclear factor κB [NF-κB]), the internal ribosome entry sequence (IRES) derived from encephalomyocarditis virus, the DNA-binding domain fusion (ZFHD1 and 3 copies of human FKBP12), and the final intron and 3′ UTR of the rabbit β-globin (RBG) gene. The FRAP*-p65 and ZFHD1-3xFKBP fusion proteins both contain an amino-terminal epitope tag from influenza virus hemagglutin (HA tag) and a nuclear localization signal (NLS) from SV40 large T antigen. AAV-CMV-TF1Nc (C) is identical to AAV-CMV-TF1 except that the HA tags are eliminated, each SV40 NLS is replaced by an NLS from human c-myc, a chimeric intron is inserted downstream of the transcription start site, and the 3′ UTR is derived from the human growth hormone (hGH) gene. In AAV-CMV-TF-rhEpo2.3 (D), elements of the above vectors were combined as indicated in “Materials and methods.” ITR indicates inverted terminal repeat sequences of AAV2.