FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia
J. Clin. Invest. Hana Raslova, et al. 114:77 doi:10.1172/JCI21197 [Go to this article.]

Figure 2
Construction and validation of a lentiviral vector encoding FLI1 cDNA. (A) Construction of a lentiviral expression vector coding for FLI1. FLI1 expression was driven by the PGK promoter. (B) Immunoblot analysis of FLI1 protein in HEL cells expressing endogenous Fli1 (lane 1), in 293T cell line transiently transfected with either the control lentiviral vector (lane 2) or with the lentiviral vector encoding FLI1 cDNA (lane 3). Five hundred × 10³ cells were loaded in lane 1 and 50 × 10³ in lanes 2 and 3. (C) Single-cell RT-PCR detection of eGFP in transduced CD34⁺ cells. Peripheral blood CD34⁺ cells obtained from healthy individuals were investigated for the presence of eGFP 6 days after infection with PGK-Fli1-IRES-eGFP virus. β2-M was used as an internal control of mRNA integrity and cell sorting. Safety control for the experiment was performed in the absence of sorted cells (lane 2: Control). (D) Immunolabeling and flow-cytometry analysis of CD34⁺ cells transduced with the lentiviral vector. CD34⁺ cells were stained with anti–CD42-PE and anti–CD41-APC Ab’s 9 days after transduction with the control lentivirus vector (dotted line) or with the FLI1 encoding lentivirus vector (thin line). Analysis of CD42 expression was performed in the cell population expressing high level of CD41 (MKs).