1. Volume 17, Issue 11, Pages 1904-1909 (November 2009)
Transgene Expression Levels Determine the Immunogenicity of Transduced Hematopoietic Grafts in Partially Myeloablated Mice 
Herena Eixarch, Alba Gómez, Elisabeth Kádár, Mónica George, Nuria Martínez, Carmen Espejo, Jordi Pétriz, Ramon Gimeno, Jordi Barquinero 
Molecular Therapy 
Volume 17, Issue 11, Pages (November 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Retroviral vectors. A bicistronic and a monocistronic vector driving EGFP expression were used. In the bicistronic vector, EGFP is placed after an IRES sequence while LTR promoter directly drives EGFP expression in the monocistronic vector. EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; LTR, long-terminal repeat.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
LTR drives EGFP expression at higher levels than IRES in BMC. Dot plots from representative samples of nontransduced BMC (left), BMC transduced with the high-EGFP (LTR) vector (center), and BMC transduced with the low-EGFP (IRES) vector (right). Plots show green fluorescence in events after appropriate gating on forward and side scatter parameters and 7-AAD labeling for dead cell exclusion. Note the different levels of EGFP expression driven by the two retroviral vectors. 7-AAD, 7-amino-actinomycin D; BMC, bone marrow cells; EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; LTR, long-terminal repeat.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Transgene-expressing cells are absent in most mice transplanted with high-EGFP grafts. Four to six weeks after transplantation (a) total donor chimerism and the proportion of EGFP+ cells (b) from mice of the four experiments were analyzed by flow cytometry. (c) Pies represent the proportion of mice lacking detectable EGFP+ cells (in black) in PB (or in BM when data was available) and those with engraftment of EGFP+ cells (in white). Among mice conditioned with busulfan, risk of clearing EGFP+ cells was significantly higher in those receiving the high-EGFP than in those receiving the low-EGFP BMC. Mice with graft failure (defined as an absence of detectable donor cells in the hematopoietic tissues analyzed) were not included in the analysis. BMC, bone marrow cells; EGFP, enhanced green fluorescent protein; PB, peripheral blood; TBI, total body irradiation.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Humoral response to EGFP. (a) Anti-EGFP Ab titers were analyzed in the sera of mice at different times after bone marrow transplantation (BMT) in the different groups. (b) Mice that specifically lost EGFP-expressing cells (rejecting) and those with EGFP-transduced donor cells (not rejecting) from the different experiments were pooled to elucidate whether the immune response toward EGFP correlated with the absence of transduced cells. Ab, antibodies; EGFP, enhanced green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
High-EGFP BMC induce cellular immune responses. (a) For the ELISPOT assays, mice were killed 10 days after transplantation and total splenocytes were assessed for IFN-γ production upon exposure to EL4-EGFP cells. Splenocytes of mice receiving high-EGFP BMC produced significantly more spots upon antigen exposure than their low-EGFP counterparts. (b) For the CTL assays we used thawed splenocytes from mice killed 30 days after BMT and EL4-EGFP cells as targets. Specific killing was evaluated by flow cytometry. Effector:target (E:T) ratios and their corresponding P values are displayed. CTL, cytotoxic T lymphocyte; EGFP, enhanced green fluorescent protein; ELISPOT, enzyme-linked immunospot assay; IFN-γ, interferon-γ; MNC, mononuclear cells; NTd, not transduced.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions