1. Transactivator and Structurally Optimized Inducible Lentiviral Vectors
Karin Haack, Adam S. Cockrell, Hong Ma, David Israeli, Steffan N. Ho, Thomas J. McCown, Tal Kafri 
Molecular Therapy 
Volume 10, Issue 3, Pages (September 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
. HIV-1 SIN vectors. Vectors are depicted as the proviral DNA form. To the right of each vector are the descriptive names (for example SV1G-tTA and SV1L-tTA) followed by the number assigned to the corresponding plasmid (continuing with the previous example, TK433 and TK434, respectively). Throughout this article all vectors are referred to by their descriptive name containing the following abbreviations: SV (single vector system), BV (binary vector system), 1 (first generation), 2 (second generation), tTA (tetracycline repressor fused to VP16 transactivator), tT65 (tetracycline repressor fused to p65 activation domain), G (GFP transgene), L (luciferase (Luc) transgene), Δatg (deletion of ATG start codon in tTA), Δwpre (deletion of the WPRE), MyoDA (MyoDA transgene). All vectors harbor a central polypurine tract (cPPT) and a WPRE, with the exception of SV1G-tTA-Δwpre and SV1L-tTA-Δwpre, which have the WPRE deleted. (A) First-generation single vector systems comprise 5′ and 3′ LTRs harboring a SIN mutation in the U3 region; a CMV promoter driving expression of the transactivator (tTA or tT65), excluding the SV1G-tTA Δatg and SV1L-tTA Δatg, which have the ATG deleted; and a TRE promoter driving expression of a transgene (GFP or Luc). (B) Binary vector systems contain a TRE-reporter and CMV-transactivator cassette on individual plasmids. The first-generation reporter cassette (BV1G or BV1L) contains the TRE directly 5′ of the reporter, whereas the second-generation reporter cassette contains the TRE in the U3 region (BV2G or BV2L). Vectors harboring the transactivator cassettes (BV1tTA or BV1tT65) are the same for both generations of binary vectors. (C) The second-generation single vectors contain the CMV-transactivator cassette 3′ of the TRE-reporter cassette within the same vector. Furthermore, the TRE has been relocated to the U3 region promoting expression of a transgene (GFP, Luc, or MyoDA).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Characterization of the Tet-inducible system in 293T cells. 293T cells were transduced with first-and second-generation, single and binary vector systems at 70 ng p24/106 cells. Transduced cells were maintained in the presence (uninduced) or absence (induced) of 1 μg/ml Dox. 293T cells were measured for expression of GFP or luciferase 10 days after withdrawal of Dox. Cells were examined for GFP fluorescence intensity by FACScan analysis as described previously [11]. Luciferase expression was analyzed from cell lysates as described previously [2]. Lysates of each sample were measured in an AutoLumat LB953 luminometer. Luciferase measurements were normalized against total protein concentration acquired for each lysate and recorded as relative light units (RLU)/μg total protein (left bottom) or as fold inducible levels (ratio of RLU/μg protein in the absence versus the presence of Dox) (right bottom). (A) 293T cells were transduced with the indicated first- or second-generation tTA-dependent vector system, cultured in the presence or absence of Dox, and analyzed for GFP expression by FACS (top) or for luciferase expression (bottom). Error bars for luciferase levels are displayed as ±SD. (B) The results are depicted for first- or second-generation tT65-dependent vector systems as described in (A) for the tTA-dependent vector systems. All experiments were performed at least three times.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Analysis of GFP expression products in 293T cells. 293T cells were transduced as described for Fig. 2. Cell extracts were prepared 10 days after withdrawal of Dox. (A) Northern blot analysis was performed on total RNA extracted from each sample. Amount of total RNA loaded for each sample was as follows: 30 μg for untransduced cells, 25 μg SV1G-tTA, 10 μg BV1G+BV1tTA, and 10 μg SV1G-tT65. The membranes were hybridized with a 32P-labeled GFP probe. The expected migration of the 3.6-kb mRNA is indicated by the open double arrow, and the 2.2-kb mRNA is indicated by the closed arrow. (B) Western blot analysis was performed on lysates from transduced 293T cells. The following amounts of protein extract were examined: 20 μg total protein for untransduced cells, 30 μg SV1G-tTA, 15 μg BV1G+ BV1tTA, 100 μg SV2G-tTA, and 7.5 μg SV1G-tT65. Blots were probed with a primary mouse anti-GFP antibody, followed by a secondary goat anti-mouse antibody conjugated to HRP. (C) Western blot analysis was performed on lysates from transduced 293T cells. The following amounts of protein extract were examined: 20 μg total protein for untransduced cells and 30 μg SV1G-tTA. Blots were probed with a primary mouse anti-GFP antibody or rabbit anti-VP16 antibody, followed by a secondary goat anti-mouse or anti-rabbit antibody conjugated to HRP, respectively. The expected migration of the GFP protein product (∼30 kDa) is indicated by the closed double arrow, and the tTA-GFP fusion protein (∼80 kDa) is indicated by the open double arrow. The tTA protein product is indicated by the closed arrowhead.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Comparison of tTA and tT65 transactivators in various cell types transduced with a second-generation binary vector system. Reporter cells were established for all cell types via transduction with the BV2L reporter vector and subsequently transduced with the corresponding transactivator vector, BV1tTA (tTA) or BV1tT65 (tT65). Luciferase expression was analyzed as described for Fig. 2. RLU/μg total protein (top) or fold inducible levels (ratio of RLU/μg protein in the absence versus the presence of Dox) (bottom) is shown ±SD. All experiments were performed at least three times.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
In vivo effects of vectors administered to the striatum of rat brains. Vectors were injected into the striatum of rat brains in the presence (I–P) or absence (A–H) of Dox. Four weeks after injection rat brains were fixed and monitored for GFP expression by fluorescence microscopy. Rats were treated with single-vector systems (SV1G-tTA (A, I), SV1G-tT65 (B, J), SV2G-tTA (C, K), SV2G-tT65 (D, L)) and binary vector systems (BV1G + BV1tTA (E, M), BV1G + BV1tT65 (F, N), BV2G + BV1tTA (G, O), BV2G + BV1tT65 (H, P)). Original magnifications of rat brain sections were 100 × in the presence or 40 × in the absence of Dox.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Differentiation of pluripotent mouse embryonic fibroblasts (MEFs) through Dox-dependent regulation of MyoDA expression. Mouse EF cells were transduced twice with the SV2MyoDA-tT65 vector to generate the MEF/SV2MyoDA-tT65 cells. Induction of myotube formation was assayed in the presence (A, C) or absence (B, D) of 1 μg/ml Dox in cells cultured under normal serum conditions (10% FBS; A, B) or minimal serum conditions (5% horse serum; C, D). Myotube formation was observed by phase-contrast microscopy. MEF/SV2MyoDA-tT65 cells were examined for expression of MyoDA, desmin, and myogenin 3, 6, or 10 days following removal of Dox and change of medium to 5% horse serum. On days 3, 6, and 10 immunofluorescence staining was performed using antibodies against MyoDA (FITC; green, E –M), desmin (TRITC; red, E, G, I, L), and myogenin (TRITC; red, F, H, K, M). Controls were cultured in the presence of 1 μg/ml Dox (L, M). The corresponding phase-contrast microscopy images are depicted to the right of the respective fluorescent image (E′ –M′).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions