1. Volume 3, Issue 2, Pages 149-159 (February 2001)
Conditional Abatement of Tissue Fibrosis Using Nucleoside Analogs to Selectively Corrupt DNA Replication in Transgenic Fibroblasts 
Masayuki Iwano, Andreas Fischer, Hirokazu Okada, David Plieth, Chengsen Xue, Theodore M. Danoff, Eric G. Neilson 
Molecular Therapy 
Volume 3, Issue 2, Pages (February 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Gancyclovir sensitivity of fibroblasts and epithelium containing FSP1.TK(ΔTK) minigenes in culture. (A) FSP1.TK minigene construction. FSP1.TK(ΔTK) constructs were assembled as described in the gene map; B, BamHI; N, NcoI; E, EcoRI; No, NotI; H, HindIII; Nh, NheI; arrows, PCR primer regions used to truncate the TK coding sequence to ΔTK. (B) 3T3 fibroblasts and MCT tubular epithelium stably transfected with FSP1.TK or control plasmids. Each cell line was cultured at a density of 103/200 μl DMEM supplemented with 10% FCS in 96-well flat-bottom plates for 24 h. After confirmation of cell adhesion to the bottom of the plates, the medium was exchanged to 10% FCS-containing DMEM with increasing concentrations of GCV (0.001 to 1 mM) and cell viability was determined 5 days later. Only the 3T3-TK fibroblasts were sensitive to GCV treatment: ○, 3T3-TK fibroblasts; ▴, MCT-TK tubular epithelium; •, 3T3-control fibroblasts; ▪, MCT-control tubular epithelium. (C) Ear fibroblasts and tubular epithelial cells were obtained from M31 FSP1.ΔTK mice and negative littermates. Primary fibroblasts and tubular epithelial cells from M31 mice were cultured at a density of 5 × 103/200 μl DMEM supplemented with 10% FCS in 96-well flat-bottom plates for 24 h. After confirmation of cell adhesion to the bottom of the plates, the medium was exchanged to 10% FCS-containing DMEM with increasing concentrations of GCV (0.01 to 100 mM). Five days later, cell viability was measured using the MTT assay. Only the fibroblasts obtained from FSP1.ΔTK mice were sensitive to GCV treatment; ○, tubular epithelial cell from FSP1.ΔTK mice; □, tubular epithelial cell from negative littermates; •, ear fibroblasts from FSP1.ΔTK mice; ▪, ear fibroblasts from ΔTK-negative littermates.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Transgenic FSP1.ΔTK cells and kidney tissue. (A) FSP1.ΔTK+ fibroblasts cultured in 0.01 mM GCV. (B) FSP1.ΔTK+ fibroblasts cultured in 10 mM GCV. After 5 days there was a marked depletion of fibroblasts grown in 10 mM GCV compared to 0.01 mM GCV. (C) Interstitial FSP1 fibroblasts stained in kidney tissue with rabbit anti-FSP1 antibody from FSP1.ΔTK− mice (arrowheads). (D) Interstitial FSP1 fibroblasts stained in kidney tissue with anti-FSP1 antibody from FSP1.ΔTK+ mice (arrowheads). (E) No reaction product in FSP1.ΔTK− renal interstitial fibroblasts stained with rabbit anti-TK antibody. (F) Interstitial fibroblasts in normal renal tissue from FSP1.ΔTK+ mice stained with anti-TK antibody (arrowheads). (G) Interstitial fibroblasts in normal renal tissue from FSP1.ΔTK+ mice stained red with anti-TK antibody (arrowheads) and compared to the same tissue double stained (H) with anti-TK antibody (red) and anti-FSP1 antibody (green), producing a yellow reaction product in fibroblasts on merging the two colors.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Differential DNA and mRNA profiles in cells and tissue harvested from FSP1.ΔTK+ and FSP1.ΔTK− mice. (A) Southern blot from tail DNA from the FSP1.ΔTK+ M31 transgenic line and a FSP1.ΔTK− (WT) littermate. The M31 line harbors six to nine copies of the FSP1.ΔTK transgene. (B) Renal tubular epithelium and ear fibroblasts were harvested from FSP1.ΔTK+ (TK+) and FSP1.ΔTK− (TK−) mice. mRNA from each cell type was reverse transcribed into cDNA with oligo(dT). After PCR for ΔTK and GAPDH, amplicons were run in agarose and stained with ethidium bromide. (C) Northern analysis of total RNA from FSP1.ΔTK+ (TK+) and FSP1.ΔTK− (TK−) kidneys was performed using 20 μg of RNA extracted from obstructed and contralateral kidneys from mice treated with GCV. After blotting, levels of mRNA encoding FSP1 were analyzed by hybridization using 32P-labeled probe for FSP1 followed by reprobing with 32P-labeled probe for GAPDH. Levels of mRNA encoding FSP1 were markedly decreased in the obstructed kidney from FSP1.ΔTK+ mice treated with GCV.TK+/TK− control, contralateral kidney mRNA from FSP1.ΔTK+ mice treated with GCV (left lane of doublet) and mRNA from FSP1.ΔTK− littermates treated with GCV (right lane of doublet). TK− + GCV, mRNA from obstructed FSP1.ΔTK− kidney following treatment with GCV.TK+ + GCV, mRNA from obstructed FSP1.ΔTK+ kidney following treatment with GCV. (D) Total RNA harvested from kidneys was tested for mRNA transcripts encoding collagen type I by cDNA amplification. In the first doublet, reaction products are from obstructed kidneys from FSP1.ΔTK+ mice following treatment with GCV; in the second set of doublets, reaction products are from contralateral, normal kidneys from obstructed FSP1.ΔTK+ mice following treatment or not with GCV; in the third set of doublets, reaction products are from obstructed kidneys from FSP1.ΔTK− mice following treatment or not with GCV.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Quantitative assessment of FSP1+ renal fibroblasts, extent of kidney fibrosis, and degree of type I collagen deposition in FSP1.ΔTK+ (TK+) and FSP1.ΔTK− (TK−) mice following treatment with GCV. (A) Evaluation of numbers of FSP1+ fibroblasts area by immunostaining following UUO. The number of FSP1+ cells was determined in mice with and without ureteral obstruction. After treatment with GCV or drug vehicle for 10 days, both obstructed and contralateral kidneys were removed, fixed in 4% buffered paraformaldehyde, and embedded in paraffin. Immunohistochemistry was performed using ABC-peroxidase-labeled rabbit anti-FSP1 antibody followed by counterstaining with Weigert's iron hematoxylin and aniline blue. The number of FSP1+ cells was counted in each of 10 nonoverlapping fields under 200× magnification and averaged. The number of FSP1+ fibroblasts was normal low (<10/200× field) in contralateral kidneys and there was no significant difference between the three groups (left three lanes). A significant reduction in numbers of FSP1+ fibroblasts (and the area of counterstain) was observed in the obstructed kidney of FSP1.ΔTK+ mice treated with GCV (TK+ + GCV) compared to FSP1.ΔTK− mice treated with GCV (TK− + GCV) or FSP1.ΔTK+ and FSP1.ΔTK−mice treated with vehicle alone (TK+/TK− – GCV); three right lanes. (B) Evaluation of renal fibrosis area by morphometric analysis following UUO. Renal interstitial fibrosis was induced by UUO in FSP1.ΔTK+ and FSP1.ΔTK−mice. After treatment with GCV or drug vehicle for 10 days, both obstructed and contralateral kidneys were removed, fixed in 4% buffered paraformaldehyde, and embedded in paraffin. The percentage fractional area of fibrosis was evaluated using Masson's trichrome staining. Morphometric analysis was performed using a standard point-counting method. The area of renal fibrosis was appropriately at background in contralateral kidneys and there was no significant difference between the three groups (left three lanes). A significant reduction in the area of renal fibrosis was observed in the obstructed kidney of FSP1.ΔTK+ mice treated with GCV (TK+ + GCV) compared to FSP1.ΔTK− mice treated with GCV (TK− + GCV) or FSP1.ΔTK+ and FSP1.ΔTK− mice treated with vehicle alone (TK+/TK− – GCV); three right lanes. (C) Evaluation of collagen type I staining by morphometric analysis following UUO. Renal interstitial fibrosis was induced by UUO in FSP1.ΔTK+ and FSP1.ΔTK− mice. After treatment with GCV or drug vehicle for 10 days, both obstructed and contralateral kidneys were removed, fixed in 4% buffered paraformaldehyde, and embedded in paraffin. Immunohistochemistry was performed using ABC-peroxidase-labeled rabbit polyclonal antibody against mouse type I collagen followed by counterstaining with hematoxylin. The area of collagen type I deposition was calculated using a standard point counting method. The area of type I collagen deposition was appropriately at background in contralateral kidneys and there was no significant difference between the three groups (left three lanes). A significant reduction in the area of collagen type I deposition was observed in the obstructed kidney of FSP1.ΔTK+ mice treated with GCV (TK+ + GCV) compared to FSP1.ΔTK− mice treated with GCV (TK− + GCV) or FSP1.ΔTK+ and FSP1.ΔTK− micetreated with vehicle alone (TK+/TK− – GCV); three right lanes.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Qualitative assessment of numbers of FSP1+ fibroblasts, area of fibrosis, and degree of type I collagen deposition in kidneys following ureteral obstruction. (A) Contralateral (normal) kidney from FSP1.ΔTK+ mice treated for 10 days with vehicle and stained to detect FSP1+ fibroblasts (arrowheads). There are a few scattered fibroblasts in the intertubular spaces forming the interstitium staining for FSP1 and no epithelium (original magnification 200x). (B) Contralateral (normal) kidney from FSP1.ΔTK+ mice treated for 10 days with vehicle and stained to detect type I collagen (arrowheads). There is a thin delicate wisp of interstitial collagen between tubules (original magnification 400x). (C) Obstructed kidney from FSP1.ΔTK+ mice treated for 10 days with vehicle and stained to detect FSP1+ fibroblasts (arrowheads). The number of FSP1+ fibroblasts is markedly increased and the increased degree of counterstained interstitium delineates the new area of fibrosis (original magnification 200×). (D) Obstructed kidney from FSP1.ΔTK+ mice treated for 10 days with vehicle and stained to detect type I collagen (arrowheads). The extent of type I collagen deposited in the interstitium has markedly increased over 10 days (original magnification 400×). (E) Obstructed kidney from FSP1.ΔTK+ mice treated for 10 days with GCV and stained to detect FSP1+ fibroblasts (arrowheads). GCV treatment has dramatically reduced the number of FSP1+ fibroblasts and the counterstained interstitial areas of fibrosis are much less compared to C (original magnification 400×). (F) Obstructed kidney from FSP1.ΔTK+ mice treated for 10 days with GCV and stained to detect type I collagen (arrowheads). GCV treatment has dramatically reduced the extent of type I collagen deposition compared to D (original magnification 400x).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions