Takiguchi, M., James, C., Josefsson, E. C., Carmichael, C. L., Premsrirut, P. K., Lowe, S. W., Hamilton, J. R., Huang, D. C. S., Kile, B. T., Dickins, R. A. (December 2010) Transgenic, inducible RNAi in megakaryocytes and platelets in mice. J Thromb Haemost, 8 (12). pp. 2751-6. ISSN 1538-7836 (Electronic) 1538-7836 (Linking)
Abstract
Summary 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.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi organism description > animal > mammal > rodent > mouse bioinformatics > genomics and proteomics > genetics & nucleic acid processing > transgenic animal |
| CSHL Authors: | |
| Communities: | CSHL Cancer Center Program > Cancer Genetics CSHL Cancer Center Shared Resources > Animal Services CSHL Cancer Center Shared Resources > DNA Sequencing Service CSHL Cancer Center Shared Resources > Flow Cytometry Service CSHL Cancer Center Shared Resources > Gene Targeting Service CSHL labs > Lowe lab CSHL Cancer Center Shared Resources > Functional Genomics and Genetics Service |
| Depositing User: | CSHL Librarian |
| Date: | December 2010 |
| Date Deposited: | 19 Oct 2011 16:45 |
| Last Modified: | 30 Oct 2015 20:44 |
| PMCID: | PMC3285240 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/15539 |
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