Figure 2

Viral vectors and promoters. (A) Provirus of the retroviral SIN-vector, ES.1 [32], with deleted viral enhancers (U3), contains enlarged packaging region (psi, psi+) and pol/env fragment harboring the native splice acceptor (SA). The tet-regulatory expression unit is inserted in sense orientation (+strand) followed by the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE, [33]). The tet-responsive promoter, Ptet-T6, was functionally coupled to the dual reporter gene lmg*, for simultaneous determination of luciferase and GFP activities. ES.1-scT6 viral vector contains an inverted tet-regulatory expression unit (−strand), transcripts were terminated at the SV40 pA site that was fused to the constitutive transport element of SRV-1 (cte, [30]). The MOV backbone is identical with ES.1-scT6, but contained a sense (+strand) insertion of the PGK- or cA-promoter driven M2 transactivator [2]. The woodchuck hepatitis virus posttranscriptional regulatory element (pre*) differs in length from the ES.1 version. (B) Outline of the weak constitutive (“c“) but tet-inducible (“A“) cA-promoter. The CAAT box of MoMuLV was added to a HIV-1 derived minimal promoter −77/+77, containing 3 SP-1 sites and the TATA-box. Positions are numbered relative to the transcriptional start site. (C) The tet-responsive minimal promoter (Ptet) used in this study, Ptet-T6, consists of a synthetic minimal promoter with consensus TATA-box and TFIIB binding site, the CMV initiator element and a TYMV 5´-UTR assembled with a tet-operator heptamer with core-spacing of 36 nt [31]. Specific restriction sites are indicated.