Journal
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Volume 8, Issue 12, Pages 2751-2756Publisher
WILEY
DOI: 10.1111/j.1538-7836.2010.04077.x
Keywords
megakaryocyte; mouse; platelet; RNA interference; tetracycline
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Funding
- National Health and Medical Research Council of Australia [516725, 575535, 461221, 361646]
- Leukemia and Lymphoma Society [SCOR 7413-07]
- European Molecular Biology Organization
- Leukaemia Foundation of Australia
- Sylvia and Charles Viertel Charitable Foundation
- Victorian State Government
- Victorian Endowment for Science, Knowledge and Innovation (VESKI)
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Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L), a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L), clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.
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