This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 27/10/2266    most recent ATVBAHA.107.149872v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohmori, T. Articles by Sakata, Y. Search for Related Content PubMed PubMed Citation Articles by Ohmori, T. Articles by Sakata, Y. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2266.)
© 2007 American Heart Association, Inc.

Thrombosis

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

Tsukasa Ohmori; Yuji Kashiwakura; Akira Ishiwata; Seiji Madoiwa; Jun Mimuro; Yoichi Sakata

From the Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan.

Correspondence to Tsukasa Ohmori, MD, PhD or Yoichi Sakata, MD, PhD, Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail tohmori{at}jichi.ac.jp or yoisaka@jichi.ac.jp.

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 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 IIb caused a significant reduction of integrin IIbβ3 (IIbβ3) expression in GFP-positive platelets. It also inhibited IIbβ3 activation assessed by the binding of JON/A, an antibody that recognizes activated IIbβ3. Talin-1 silencing by the same method resulted in normal IIbβ3 expression but deficient inside-out IIbβ3 signaling.

Conclusions— shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.

The expression of short hairpin RNA (shRNA) in hematopoietic stem cells by a lentiviral vector resulted in inhibition of targeted protein in platelets, suggesting that shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. Talin silencing by this method caused significant reduction of inside-out IIbβ3 signaling in platelets.

Key Words: shRNA • RNA interference • platelets • talin • integrin

This article has been cited by other articles:

				L. Gilles, D. Bluteau, S. Boukour, Y. Chang, Y. Zhang, T. Robert, P. Dessen, N. Debili, O. A. Bernard, W. Vainchenker, et al. MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9 Blood, November 5, 2009; 114(19): 4221 - 4232. [Abstract] [Full Text] [PDF]