TY - JOUR
T1 - Vivo-Morpholino knockdown of alphaIIb
T2 - A novel approach to inhibit thrombocyte function in adult zebrafish
AU - Kim, Seongcheol
AU - Radhakrishnan, Uvaraj P
AU - Rajpurohit, Surendra Kumar
AU - Kulkarni, Vrinda
AU - Jagadeeswaran, Pudur
N1 - 2009 Elsevier Inc. All rights reserved.
PY - 2010/3/15
Y1 - 2010/3/15
N2 - Knockdown of protein function by antisense oligonucleotides has been used to understand the protein function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos to evaluate hemostatic function in adult animals. We injected alphaIIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte function was assayed by time to aggregation assay of the citrated blood, annexin V binding to thrombocytes, and gill bleeding. The thrombocyte functional inhibition occurred in 24 h after alphaIIb Vivo-Morpholinos injection and reached a maximum in 48 h. However, in 72 h, the inhibition was no longer observed. Reduction of annexin V binding to thrombocytes and increased gill bleeding were observed 48 h after alphaIIb Vivo-Morpholino injections. The action of the alphaIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced alphaIIb mRNA and the reduction of alphaIIb in thrombocytes of fish treated with alphaIIb Vivo-Morpholino. These results provide the first proof of principle that thrombocyte function can be inhibited by thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown thrombocyte-specific genes to conduct biochemical studies in thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit thrombocyte function in adult zebrafish.
AB - Knockdown of protein function by antisense oligonucleotides has been used to understand the protein function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos to evaluate hemostatic function in adult animals. We injected alphaIIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte function was assayed by time to aggregation assay of the citrated blood, annexin V binding to thrombocytes, and gill bleeding. The thrombocyte functional inhibition occurred in 24 h after alphaIIb Vivo-Morpholinos injection and reached a maximum in 48 h. However, in 72 h, the inhibition was no longer observed. Reduction of annexin V binding to thrombocytes and increased gill bleeding were observed 48 h after alphaIIb Vivo-Morpholino injections. The action of the alphaIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced alphaIIb mRNA and the reduction of alphaIIb in thrombocytes of fish treated with alphaIIb Vivo-Morpholino. These results provide the first proof of principle that thrombocyte function can be inhibited by thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown thrombocyte-specific genes to conduct biochemical studies in thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit thrombocyte function in adult zebrafish.
KW - Animals
KW - Blood Platelets
KW - Gene Knockdown Techniques
KW - Oligonucleotides, Antisense
KW - Platelet Aggregation
KW - Platelet Function Tests
KW - Platelet Membrane Glycoprotein IIb
KW - Zebrafish
U2 - 10.1016/j.bcmd.2009.12.004
DO - 10.1016/j.bcmd.2009.12.004
M3 - Article
C2 - 20045356
SN - 1079-9796
VL - 44
SP - 169
EP - 174
JO - Blood Cells, Molecules, and Diseases
JF - Blood Cells, Molecules, and Diseases
IS - 3
ER -