1. Volume 3, Issue 4, Pages 565-573 (April 2001)
Comparison of Five Retrovirus Vectors Containing the Human IL-2 Receptor γ Chain Gene for Their Ability to Restore T and B Lymphocytes in the X-Linked Severe Combined Immunodeficiency Mouse Model 
Guillermo J. Avilés Mendoza, Nancy E. Seidel, Makoto Otsu, Stacie M. Anderson, Karen Simon-Stoos, Adrianna Herrera, Shelley Hoogstraten-Miller, Harry L. Malech, Fabio Candotti, Jennifer M. Puck, David M. Bodine 
Molecular Therapy 
Volume 3, Issue 4, Pages (April 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Retroviral vectors compared in this study. MND-hIL2RG: Myoproliferative sarcoma virus (MPSV) long terminal repeat (LTR), splice donor (SD), splice acceptor (SA), human IL2RG cDNA; titer, ~1 × 106 infectious particles/ml. GC-hIL2RG: Moloney murine leukemia virus LTR, SA, SD, human IL2RG cDNA; titer, ~1 × 105 infectious particles/ml. MFG-S-hIL2RG: MPSV LTR, SA, SD, human IL2RG cDNA; titer, ~5 × 105 infectious particles/ml. Ha−VL30-hIL2RG: Harvey murine sarcoma virus (HaSV) LTR, human IL2RG cDNA; titer, ~5 × 105 infectious particles/ml. Ha+VL30-hIL2RG: HaSV LTR, VL-30, human IL2RG cDNA; titer, ~1 × 106 infectious particles/ml.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Detection of intact proviral sequences in primary CFU-S colonies. Southern blot analysis of genomic DNA samples isolated from macroscopic CFU-S digested with SsiI (MND, GC, MFG-S, and Ha+) or KprI (Ha−) and hybridized with the hIL2RG cDNA. This probe recognized both the hIL2RG gene in the provirus (P) and the mouse IL2rg gene (E), which serves as an internal control. DNA from producer cell lines (+C) and untransduced NIH 3T3 cells (–C) were used as positive and negative controls. MFGs, DNA from CFU-S exposed to the MFG-S-hIL2RG vector; MND, DNA from CFU-S exposed to the MND-hIL2RG vector; GC, DNA from CFU-S exposed to the GC-hIL2RG vector; Ha–, DNA from CFU-S exposed to the Ha−-hIL2RG vector; Ha+, DNA from CFU-S exposed to the Ha+VL30-hIL2RG vector.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Northern blot analysis of hIL2RG mRNA levels in transduced primary CFU-S colonies. RNA was isolated from macroscopic CFU-S and hybridized with the hIL2RG cDNA. Membranes were stripped and reprobed with β-actin as a loading control. RNAs from a producer cell line (+C) and untransduced NIH 3T3 cells (–C) were used as positive and negative controls. The positions of the spliced and unspliced IL2RG mRNA and proviral IL2RG fragments are indicated. The exposures of the different blots are not identical. RNA samples include some from the same colonies analyzed in Fig. 2. MFGs, RNA from CFU-S exposed to the MFG-S-hIL2RG vector; MND, RNA from CFU-S exposed to the MND-hIL2RG vector; Ha+, RNA from CFU-S exposed to the Ha+VL30-hIL2RG vector; Ha–, RNA from CFU-S exposed to the Ha−VL30-hIL2RG vector; GC, RNA from CFU-S exposed to the GC-hIL2RG vector.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Southern blot analysis of proviral copy number in W/Wv or XSCID recipient mice 19 to 22 weeks after transplantation with bone marrow cells transduced with hIL2RG retrovirus vectors. Genomic DNA was extracted from bone marrow (Bm), spleen (S), thymus (T), splenic B cells (B), and lymph nodes (L) and digested with SstI (MND, GC, MFG-S, and Ha+) or KpnI (Ha−) and hybridized with the hIL2RG cDNA. This probe recognized both the hIL2RG gene in the provirus (P) and the mouse IL2rg gene (E), which serves as an internal control. DNA from producer cell lines (+C) and untransduced NIH 3T3 cells (3) were used as positive and negative controls. MFGs, DNA from tissues derived from cells exposed to the MFG-S-hIL2RG vector; MND, DNA from tissues derived from cells exposed to the MND-hIL2RG vector; GC, DNA from tissues derived from cells exposed to the GC-hIL2RG vector; Ha-, DNA from tissues derived from cells exposed to the Ha−-hIL2RG vector; Ha+, DNA from tissues derived from cells exposed to the Ha+VL30-hIL2RG vector.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Northern blot analysis of hIL2RG mRNA levels in tissues from W/W or XSCID recipient mice 19 to 22 weeks after transplantation with bone marrow cells transduced with hIL2RG retrovirus vectors. RNA was isolated from bone marrow (Bm), spleen (S), thymus (T), and splenic B cells (B) and hybridized with the hIL2RG cDNA. Membranes were stripped and reprobed with β-actin as a loading control. The exposures of the different blots are not identical. The RNA samples include some from the same tissues analyzed in Fig. 4. MFGs, RNA from tissues derived from cells exposed to the MFG-ShIL2RG vector; MND, RNA from tissues derived from cells exposed to the MND-hIL2RG vector; Ha+, RNA from tissues derived from cells exposed to the Ha+VL30-hIL2RG vector; Ha-, RNA from tissues derived from cells exposed to the Ha−VL30-hIL2RG vector; GC, RNA from tissues derived from cells exposed to the GC-hIL2RG vector.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Comparison of human IL2RG mRNA levels in hematopoietic tissues of W/Wv mice repopulated with wild-type cells transduced with the five different retrovirus vectors. The hIL2RG mRNA and proviral copy number was determined by Northern and Southern blot, respectively. Averages and SD of relative hIL2RG mRNA levels are shown. Each value represents the mean of at least five animals analyzed for each vector. RNA from a 3T3 cell line containing a single copy of a Ha+VL30-hIL2RG retrovirus was used as a standard for normalization. The MFG-S vector expressed significantly more hIL2RG mRNA than any other vector (P < 0.01), and the Ha+VL30 vector expressed significantly less hIL2RG mRNA than any other vector (P < 0.01). The differences between the levels of hIL2RG mRNA in cells containing the Ha-, GC, and MND vectors were statistically significant in all cases (P < 0.04).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Flow cytometric analysis of human 7c expression in peripheral blood lymphocytes from W/W mice repopulated with wild-type C57BL/6 bone marrow cells transduced with the MND- (left) or MFG-S-hIL2RG (right) vector. The x axis represents cells stained with PE-conjugated anti-TUGh4 while the y axis represents cells stained with FITC-conjugated anti-CD8, anti-CD4, or anti-B220. The analysis was conducted 18–24 weeks posttransplantation on cells in the lymphocyte gate.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
Flow cytometric analysis of human γc expression on TUGh4+ peripheral blood lymphocytes from W/Wv mice repopulated with mIL2RG-deficient C57BL/6 –/Y bone marrow cells transduced with the MND- (left) or MFG-S-hIL2RG (right) vector. (Top) The analysis was conducted on cells in the TUGh4+ gate. The x axis represents cells stained with PE-conjugated anti-TUGh4 while the y axis represents cells stained with FITC-conjugated anti-B220. (Bottom) The analysis was conducted on cells in the TUGh4+ gate. The x axis represents cells stained with PE-Cy-5-conjugated anti-B220, while the y axis represents cells stained with FITC-conjugated anti-mouse IgM. The analysis was conducted 18–24 weeks posttransplantation as in Fig. 7.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions