1. Volume 10, Issue 4, Pages 758-767 (October 2004)
The human SCGB2A2 (Mammaglobin-1) promoter/enhancer in a helper-dependent adenovirus vector directs high levels of transgene expression in mammary carcinoma cells but not in normal nonmammary cells 
Chang-Xin Shi, Michael A. Long, Limin Liu, Frank L. Graham, Jack Gauldie, Mary M. Hitt 
Molecular Therapy 
Volume 10, Issue 4, Pages (October 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Analysis of the activity of SCGB2A2 promoter fragments using a plasmid transfection system. Cells were transfected with plasmids carrying the luciferase gene under the control of various size fragments of the SCGB2A2 promoter as indicated and a second plasmid carrying lacZ under the control of the cytomegalovirus (CMV) immediate early promoter to normalize for transfection efficiency. Cells were harvested and assayed 48 h later for luciferase and β-galactosidase expression as described under Materials and Methods. No promoter was inserted upstream of luciferase in the basic plasmid. Values represent the means ± the standard deviations of activities in duplicate transfections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Activity and specificity of fragments of the SCGB2A2 promoter delivered by helper-dependent adenovirus vectors. Luciferase activity was measured in human BrCa (T47D) or normal human fibroblasts (MRC5) 48 h post-infection with HDAd vectors carrying the indicated fragments of the SCGB2A2 promoter, at an m.o.i. of 2000 particles per cell (equivalent to 20–50 pfu per cell with a first-generation vector). Values represent the means ± the standard deviations of activities in triplicate infections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Deletion analysis of the SCGB2A2 promoter. The structures of the SCGB2A2 promoter fragments used in this experiment are shown at the top. Sequences carried by the HDAd vectors are indicated by bold arrows or bars. Deleted sequences are indicated by a thin line. Nucleotides are numbered relative to the translational start site of the SCGB2A2 gene. Results of the expression analysis are shown below. Luciferase expression was determined in human T47D BrCa cells (black) or normal human MRC5 fibroblasts (white) 48 h post-infection with HDAd vectors encoding the luciferase gene driven by SCGB2A2 promoter fragments as indicated. Values represent the means ± the standard deviations of activities in triplicate infections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
The effects of deletion of the potential enhancer(s) between 4.4 and 7 kb of the SCGB2A2 promoter. The structures of the SCGB2A2 promoter fragments used in this experiment are shown on top, and the results of the expression analysis are shown below. SCGB2A2 sequences carried by the HDAd vectors are indicated by bold arrows or bars. Deleted sequences are indicated by a thin line. Nucleotides are numbered relative to the translational start site of the SCGB2A2 gene. Below, luciferase expression was determined in human T47D BrCa cells (black) or normal human MRC5 fibroblasts (white) 48 h post-infection with HDAd vectors encoding the luciferase gene driven by the indicated SCGB2A2 promoter fragments. Values represent the means ± the standard deviations of activities from triplicate infections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Regulatory regions of the SCGB2A2 promoter. (A) Schematic diagram of the potential minimal SCGB2A2 promoter between 1 and 344 bp and a strong enhancer between 4.4 and 5.5 kb upstream of the SCGB2A2 coding sequence. (B) Sequence of the SCGB2A2 enhancer and potential transcription factor binding sites. Potential binding sites are underlined. −5471 to −4360 of the enhancer corresponds to to of GenBank Accession No. AP (C) Sequence of the minimal SCGB2A2 promoter and potential transcription factor binding sites. Potential binding sites are underlined. −280 to +64 corresponds to to of AP The engineered SacI site is shown in italic. The start of the open reading frame is shown in uppercase. Noncoding sequences are in lowercase. The enhancer and promoter sequences are numbered relative to the transcription start site.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
SCGB2A2 promoter activity in various breast cancer and normal cell lines. Indicated cell lines were infected with the SCGB2A2 promoter vector HDMP25K (black bars) or an Ad vector carrying a murine CMV-luciferase expression cassette (gray bars) and then assayed for luciferase expression 48 h later. T47D, MDA-MB468, MCF7, and MT1A2 are breast cancer cell lines. MRC5, HEK, HER, and NIH3T3 are normal cell lines × the ratio of luciferase activity in HDMP25K infections to that in AdCMV-Luc infections for each cell line is given above the gray bars. Values represent the means ± the standard deviations of activities from triplicate infections.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Activity of the human SCGB2A2 promoter in vivo in a mouse mammary carcinoma model. (A and B) Mice were injected intratumorally with HDMP25K (containing the 25-kb SCGB2A2 promoter-driven luciferase expression cassette) or with a first-generation adenovirus vector containing a murine CMV-luciferase expression cassette. (C and D) In a separate experiment, mice were injected intratumorally with an HDAd carrying a murine CMV-luciferase expression cassette. On day 2 (A and C) or day 7 (B and D) three mice in each group were euthanized, then tissues were harvested and assayed for protein and luciferase activity as described under Materials and Methods. Luciferase expression levels in HDMP25-infected tissues are shown as black bars, levels in FGAdmCMV-luciferase-infected tissues are shown as gray bars, and levels in HDAdmCMV-luciferase-infected tissues are shown as hatched bars. Values represent the means ± the standard deviations for each group; each sample was assayed in triplicate.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions