1. In vivo retroviral gene transfer by direct intrafemoral injection results in correction of the SCID phenotype in Jak3 knock-out animals
by Christine S. McCauslin, John Wine, Linzhao Cheng, Kim D. Klarmann, Fabio Candotti, Peter A. Clausen, Sally E. Spence, and Jonathan R. Keller
Blood
Volume 102(3):
August 1, 2003
©2003 by American Society of Hematology

2. Direct intrafemoral injection procedure.
Direct intrafemoral injection procedure. One or both hind limbs of anesthetized mice were injected by DII with 30 μL PKH-26 dye at a concentration of 1 × 10-5 M. Bone marrow from injected femurs was harvested and analyzed for PKH-26 labeling by fluorescence microscopy (original magnification, × 400). The data shown are representative of 3 separate experiments.
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Direct intrafemoral injection of recombinant adenovirus.
Direct intrafemoral injection of recombinant adenovirus. The rAdluc virus was produced, purified, and administered by DII according to the procedures described in “Materials and methods.” Following DII, BMCs were harvested from individual mice (1-6) and assayed for luciferase activity 24 hours after culture in vitro (A) or were harvested from mice 24 hours after DII in vivo (B). Saline injection was used as a negative control (-), and DII of femurs removed from host was used as a positive control (+). These results are representative of 3 separate experiments.
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Gene transfer to bone marrow progenitors and to peripheral blood cells 2 months after transplantation.
Gene transfer to bone marrow progenitors and to peripheral blood cells 2 months after transplantation. Retroviral vector was produced, purified, and injected into 5-FU–treated mice by DII according to the procedures described in “Materials and methods.” BMCs from saline (control) or MGIN virus–injected femurs were transplanted into primary irradiated recipient mice 24 hours after DII, and PB was analyzed 2 months after transplantation for EGFP expression by flow cytometry (A). In addition, BMCs were obtained from DII-treated 5-FU day-5 animals 24 and 48 hours following injection and plated at 5 × 104 cells per plate in soft agar in duplicate in the presence of IL-3, IL-6, SCF, and granulocyte colony-stimulating factor (G-CSF). Additionally, plating was carried out in the presence or absence of 600 μg/mL G418. Seven days after plating, viable colonies of 50 or more cells were scored. The percentage of G418-resistant colonies was calculated by dividing the number of colonies growing in the presence of G418 by the total number of colonies scored in the absence of G418 (B). The data presented are representative of 3 experiments.
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology

5. EGFP expression in hematopoietic cells in recipient mice undergoing secondary transplantation.
EGFP expression in hematopoietic cells in recipient mice undergoing secondary transplantation. C57BL/6 mice underwent DII of MGIN retrovirus and transplantation, and then EGFP+ BMCs were obtained from these mice after 6 months by FACS sorting and transplanted into secondary irradiated recipient mice as described in “Materials and methods.” Two to 5 months following secondary transplantation of EGFP+ cells, PB was analyzed for the presence of donor cells in 3 separate mice (A). (B) In vivo gene transfer to PHSCs. Four months following secondary transplantation, PB was analyzed for GFP expression in T cells (Thy 1.2), B cells (B220), and granulocytes (Gr-1) from a representative animal.
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Direct intrafemoral injection of MND.Jak3 virus into Jak3-/- mice.
Direct intrafemoral injection of MND.Jak3 virus into Jak3-/- mice. MND.Jak3 retrovirus (JAK3 RV) was administered to Jak3-/- and control mice by DII according to the procedures described in “Materials and methods.” Two months following treatment, PB cells from control C57BL/6 mice (column 1), mock-injected Jak3-/- mice (column 2), and MND.Jak3-injected mice (Jak3-/- + JAK3 RV) (column 3) were analyzed for the presence of CD8+ and CD4+/62L+ T cells (% of total population indicated in quadrant).
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Functional analysis of lymphoid cell populations following DII of Jak3-/-.
Functional analysis of lymphoid cell populations following DII of Jak3-/-. For T-cell proliferation, PB cells from control C57/BL6 mice (WT), Jak3-/- mice (KO), DII-injected Jak3-/- mice (KO + DII), or Jak3-/- mice undergoing transplantation with ex vivo–infected BMCs (Ex Vivo) were plated in the presence of either media alone or with ConA plus IL-2, cultured for a total of 48 hours in vitro, pulsed with [3H]thymidine, and then harvested according to the procedures described in “Materials and methods.” Data are plotted as the means ± SDs counts per minute (cpm) of triplicate wells (A). Error bars indicate standard deviations. (B) For B-cell function, mice were challenged with chicken IgG, and 14 days after inoculation serum plasma was collected to measure for the presence of chicken IgG–specific antibodies (absorbance at 405 nm in ELISA assay according to the procedures described in “Materials and methods”).
Christine S. McCauslin et al. Blood 2003;102:
©2003 by American Society of Hematology