1. Volume 11, Issue 4, Pages 627-637 (April 2005)
Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond– blackfan anemia using inducible expression of siRNA against RPS19 
Koichi Miyake, Johan Flygare, Thomas Kiefer, Taiju Utsugisawa, Johan Richter, Zhi Ma, Maciej Wiznerowicz, Didier Trono, Stefan Karlsson 
Molecular Therapy 
Volume 11, Issue 4, Pages (April 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Lentiviral vector expressing the siRNA against RPS19. (A) A schematic diagram of the human RPS19 gene showing the locations within the gene and the sequences of siRNA directed against RPS19 (A, B, and C) and control (Scr). Sense and antisense siRNA sequences are shown in bold and loop sequences [35] and restriction sites (BamHI and HindIII) are underlined. (B) Diagrams of the siRNA vector (LV-TH-siRNA) and the tet repressor vector (pLV-tTR-KRAB-Red) used in this study.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Inducible expression of GFP and effective downregulation of RPS19 mRNA in TF-1 and UT-7 cells. (A) Inducible expression of the GFP transgene. TF-1-B and UT-7-B cells were cultured with or without Dox (0.5 μg/ml) in the presence of GM-CSF (5 ng/ml) for 2 days. Induced expression of GFP was analyzed by flow cytometer. (B) Inducible suppression of RPS19 expression. Total RNAs were extracted from established TF-1 (left) and UT-7 (right) cell lines incubated with or without Dox. Expression of RPS19 was analyzed using the real-time Q-RT-PCR method. The bar graph shows the means of two independent experiments as a percentage of RPS19 expression compared to the expression level in mock cell lines cultured without Dox. The standard deviations are indicated as error bars.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
The RPS19 protein is downregulated in Dox-induced TF-1 and UT-7 cells. (A) Recombinant RPS19 protein with 6×His tag (lane 1) and cell lysate of TF-1 cells (lane 3) was analyzed by Western blot using the RPS19 antibody (lane 2, size marker). (B) TF-1 cells were cultured with or without Dox in the presence of GM-CSF for 5 days and analyzed by Western blot using RPS19- or actin-specific antibody. (C) Densitometry analysis of Western blots (means of two independent experiments) of established TF-1 (left) and UT-7 (right) cell lines. The bar graph shows percentage of RPS19 protein expression compared to the expression level in mock cell lines cultured without Dox. The standard deviations are indicated as error bars.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Reduced proliferation capacity in Dox-induced TF-1 and UT-7 cells. 1 × 105 cells were cultured with or without Dox in the presence of (A and B) GM-CSF or (C and D) Epo. The viable cell numbers were determined every 2 days by trypan blue staining. Each number represents the average from three independent experiments. The standard deviations are indicated as error bars. The asterisks indicate a significant difference compared to Dox (−) cells. *P < 0.05, **P < 0.01, ***P <
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Reduced hematopoietic colony formation in RPS19 siRNA-treated TF-1 and UT-7 cell lines. (A) Pictures of colonies from TF-1-B at day 14 without (left) and with (right) Dox. GFP expression was detected using an immunofluorescence microscope. 2 × 103 cells were plated in methylcellulose with or without Dox in the presence of (B and C) GM-CSF or (D and E) Epo. Colonies were counted on day 14. Each number represents the average from three independent experiments. The standard deviations are indicated as error bars. The asterisks indicate a significant difference compared to Dox (−) cells. *P < 0.005, **P < 0.05.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Cell surface maker analysis of (A) TF-1-Scr and (B) TF-1-C cells. TF-1-Scr and TF-1-C cells were cultured with or without Dox in the presence of GM-CSF. After 10 days, cells were stained with anti-CD13, CD41, or glycophorin A (GPA) and analyzed by flow cytometer. (C) DAF (2,7-diaminofluorene) staining for detection of hemoglobin. TF-1 (left) and UT-7 (right) cells were cultured with or without Dox in the presence of Epo for 7 days. The percentages of cells producing hemoglobin were estimated by DAF staining. Each number represents the average from three independent experiments. The standard deviations are indicated as error bars. The asterisk indicates a significant difference compared to Dox (−) cells. *P < 0.01.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Phenotype correction by transduction with lentiviral vector expressing modified RPS19. (A) Plasmid construction of lentiviral vector expressing YFP and modified human RPS19. The RPS19 transgene in the lentiviral construct pLV-mRIY has 24 bases changed to make it resistant to silencing by the RPS19-siRNA-B, while producing the wild-type protein. (B) Flow cytometric analysis of transduced TF-1-B cells. Two days after transduction with LV-mRIY, YFP-positive cells (boxed fractions) were sorted by FACS Vantage. L, YFP low fraction. H, YFP high fraction. (C) LV-mRIY nontransduced (NT) and transduced (Low and High) TF-1-B cells were incubated for 7 days in the presence of GM-CSF (left) or Epo (right) with or without Dox (0.5 μg/ml), after which cell growth was estimated by MTT assay. (D) Colony formation assay of LV-mRIY-transduced TF-1-B cells. 2 × 103 cells were plated in methylcellulose with or without Dox in the presence of GM-CSF (left) or Epo (right). (E) DAF staining of LV-mRIY-transduced TF-1-B cells. LV-mRIY-transduced TF-1-B cells were cultured with or without Dox in the presence of Epo for 7 days. After staining with DAF, DAF-positive blue cells were counted. Each number represents the average from two independent experiments. The standard deviations are indicated as error bars.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions