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Blood, 1 September 2005, Vol. 106, No. 5, pp. 1559-1564.
Prepublished online as a Blood First Edition Paper on May 12, 2005; DOI 10.1182/blood-2005-02-0638.
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Submitted February 15, 2005
Accepted April 5, 2005
Conditional overexpression of transgenes in megakaryocytes and platelets in vivo
Hao G Nguyen, Guangyao Yu, Maria Makitalo, Dan Yang, Hou-Xiang Xie, Matthew R Jones, and Katya Ravid*
Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA
* Corresponding author; email: ravid{at}biochem.bumc.bu.edu.
Megakaryocyte-specific transgene expression has proven valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we have employed the tetracycline/doxycycline (Tet)-off system to conditionally overexpress genes in megakaryocytes (MKs) and platelets in vivo. We generated three transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the megakaryocyte-specific, platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal CMV-tTA responsive promoter driving prokaryotic  -Galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-VP16 was crossbred to a responder line. The homozygous double transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by beta-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16 that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.
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