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Silencing of a Targeted Protein in In Vivo Platelets Using a Lentiviral Vector Delivering Short Hairpin RNA Sequence

Objective— Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets. Methods and Results— To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib&agr; promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin &agr;IIb caused a significant reduction of integrin &agr;IIb&bgr;3 (&agr;IIb&bgr;3) expression in GFP-positive platelets. It also inhibited &agr;IIb&bgr;3 activation assessed by the binding of JON/A, an antibody that recognizes activated &agr;IIb&bgr;3. Talin-1 silencing by the same method resulted in normal &agr;IIb&bgr;3 expression but deficient inside-out &agr;IIb&bgr;3 signaling. Conclusions— shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.