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.


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 alpha 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 alphaIIb caused a significant reduction of integrin alphaIIb beta3 (alphaIIb beta3) expression in GFP-positive platelets. It also inhibited alphaIIb beta3 activation assessed by the binding of JON/A, an antibody that recognizes activated alphaIIb beta3. Talin-1 silencing by the same method resulted in normal alphaIIb beta3 expression but deficient inside-out alphaIIb beta3 signaling.


CONCLUSIONS
shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.
Ohmori
Tsukasa
T
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. tohmori@jichi.ac.jp
Kashiwakura
Yuji
Y
Ishiwata
Akira
A
Madoiwa
Seiji
S
Mimuro
Jun
J
Sakata
Yoichi
Y
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D045744:Cell Line, Tumor; D048430:Cell Shape; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics; D040201:Platelet Membrane Glycoprotein IIb / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D016608:Talin / Q000235:genetics / Q000378:metabolism*; D034061:Thrombopoiesis; D014161:Transduction, Genetic
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D040201:Platelet Membrane Glycoprotein IIb; D034741:RNA, Small Interfering; D016608:Talin; C496077:Tln1 protein, mouse; D049452:Green Fluorescent Proteins; D012320:RNA Polymerase III