1. Endogenous GATA Factors Bind the Core Sequence of the tetO and Influence Gene Regulation with the Tetracycline System 
David J. Gould, Yuti Chernajovsky 
Molecular Therapy 
Volume 10, Issue 1, Pages (July 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Sequence analysis of the transcription factor binding sites within the Ptet. The sequence is divided into seven repeats of the tetO, which are underlined, and the minimal CMV promoter region, which is in italics. Transcription factor binding motifs within the Ptet were determined using the sequence analysis software MacDNASIS Pro v.3.6 (Hitachi Software Engineering America Ltd., San Bruno, CA, USA) and are indicated by annotated boxes. The restriction site StuI is also indicated.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Schematic representation of plasmids and functional expression of luciferase. (A) Diagrams of Ptet vector deletion constructs. (Solid triangle) SV40 early/late poly(A) signal, (gray box) 3′UTR; smCMV, small minimal CMV promoter from −52 to −14 of the wild-type promoter; tetO, seven repeats of the tetracycline operon; Psmtet, tetO driving the smCMV promoter; Ptet, seven repeats of the tetO driving the mCMV promoter (−52 to +77 of the wild-type promoter). (Right) Luciferase expression was assessed 24 h after transfection of DTF (0.5 × 106 cells/well) with 5 μg pGL2-Basic or equivalent molar amounts of pGsmCMV, pGtetOL, pGtL, and pGTL. (B) Comparison of regulated luciferase expression from pGtL and pGTL. DTF plated at 0.5 × 106 cells/well were transfected with pGCMV alone or cotransfected with pGtL (5 μg) or pGTL and pUHG17-1 at a ratio of 1:1, with molar equivalence of all plasmids based on the 5 μg of pGtL (the smallest vector). Transfected cells were maintained uninduced (open bars) or induced with Dox (1 μg/ml, solid bars) for 24 h. Each value is the mean luciferase measurement adjusted for protein content from triplicate transfections with vertical bars representing the SEM. Values above bars indicate the degree of induction compared with the uninduced control. Significant differences from respective uninduced controls are indicated by * (P < 0.01) and ** (P < 0.005).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
EMSA probes and binding of endogenous and overexpressed GATA factors to the tetO. (A) Oligonucleotide probes used in EMSA. Boldface indicates the locations of GATA motifs, and boxed nucleotides in the mutated probes indicate the positions of substitutions that delete the GATA site. The tetO probe contains only the palindromic region of the tetR recognition site, while the 42-bp tetO contains both motifs for the tetR recognition and the linker sequence in which the ISRE motif is located. Nuclear extracts (NE) from (B) Jurkat, (C) DTF, and (D) transfected 293T cells form DNA–protein complexes (indicated by arrows) with radiolabeled GATA (G) and tetO (T) probes.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Analysis of complexes formed with GATA factors in Jurkat and DTF nuclear extracts through competition binding. (A) Prevention of GATA factor complexes formed with Jurkat nuclear extracts through cold competition binding. NE of nontransfected Jurkat cells were incubated with radiolabeled GATA and tetO probes alone and in the presence of cold probe at 5-, 20-, and 100-fold excess. With increasing concentrations of cold probe there is a reduction in the detected amount of DNA–protein complex indicated by the arrows. (B and C) Analysis of complexes formed with GATA factors in DTF nuclear extracts through cold and cross-competition binding. (B) NE of nontransfected DTF cells were incubated with radiolabeled tetO probes alone (lane 1) or in the presence of excess unlabeled probe. Unlabeled probes were the tetO (T), mutated tetO (TM), GATA (G), and mutated GATA (GM), incubated at between 2- and 16-fold excess. (C) DTF NE incubated with labeled GATA probe and competitive binding with unlabeled tetO probe and the mutated forms of the GATA and tetO probe at an excess of between 4- and 100-fold.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Interactions between endogenous and overexpressed GATA factors with the tetO are confirmed by supershift. NE prepared from (A) Jurkat or (B) DTF cells were treated with antibody to mouse GATA-3 (G3), -4 (G4), or -6 (G6) prior to incubation with labeled tetO probe. (C) NE from 293T cells transfected with plasmid encoding mGATA-4 were incubated with antibody to mGATA-4 or mGATA-6 prior to binding with labeled GATA or tetO probe.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Binding of GATA-4 to the 42-bp tetO utilized in the Ptet. NE of transiently transfected 293T cells were used to characterize the interactions between mGATA-4 with the 42-bp tetO probe (A) in competition assays and (B) by supershift analysis. In (A), NE from pcDNA3- or pcDNAG4-transfected 293T cells were incubated with labeled GATA (G) or 42-bp tetO (Tt) probes. Competition and cross-competition were determined using the relevant unlabeled cold probe in excess as indicated. Supershift analysis (B) was utilized to confirm the interactions between mGATA-4 and the labeled Tt probe using the same antibodies as described for Fig. 5.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Comparison of binding of endogenous proteins from different cell lines to the tetO probe. (A) NE (2 μg) from Cos-7, DTF, HT 1080, and A431 cells were incubated with labeled tetO probe prior to electrophoresis; the intensity of the upper complexes (indicated by the arrow) was determined by analysis with Adobe PhotoShop v.6 and (B) plotted as a percentage.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Functional effects of GATA factor interaction with the Ptet. (A and B) Induction of Ptet with GATA factors and influence of GATA factors on Ptet induction with (C and D) rtTA and (E and F) tTA. In all experiments luciferase expression was assessed 24 h after cotransfection of DTF (0.5 × 106 cells/well; A, C, and E) and A431 (0.25 × 106 cells/well; B, D, and F) cells. Cells were transfected with a total of 15 μg of DNA consisting of pGTL (5 μg); GATA-4, GATA-5, or GATA-6 expression vector (5 μg); and pcEGFP (5 μg in A and B) or pUHG 17-1 encoding rtTA (5 μg in C and D) or pUHD 15-1 encoding tTA (5 μg in E and F). In transfections that did not include a GATA expression vector an equivalent amount of pcEGFP was included in the transfection mix. When cells were induced with Dox the concentration was 1 μg/ml. Luciferase values are the means from triplicate transfections, numbers above the bars indicate the degree of induction compared with the pGTL or uninduced control. Significant differences from these controls are indicated by * (P < 0.05), ** (P < 0.02), *** (P < 0.01), § (P < 0.002), §§ (P < ), and §§§ (P < ).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

10. Fig. 9
Inhibition of Ptet induction with rtTA by titrated expression of GATA factors. DTF (0.1 × 106 cells/well in 12-well plates) were cotransfected with 25 ng pGTL, 125 ng molar equivalent of pUHG 17-1 encoding the rtTA, and GATA-factor-encoding vector at ratios of 0.1:1 (12.5 ng), 1:1 (125 ng), and 10:1 (1250 ng) with respect to pUHG The vector pRL-CMV (100 ng) was included in all transfections for standardization of transfection efficiency and the total amount of DNA in all transfections was made up to the molar equivalent of 1.5 μg of pGTL with the plasmid pcDNA3. For induction of gene expression Dox (1 μg/ml) was added to the cells for 24 h. Luciferase values were normalized relative to the Renilla luciferase activity. Significant differences from the pGTL + pUHG Dox value are indicated by * (P < 0.05) and ** (P < 0.01).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions