Transgenic, inducible RNAi in megakaryocytes and platelets in mice

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)

URL: http://www.ncbi.nlm.nih.gov/pubmed/21138522
DOI: 10.1111/j.1538-7836.2010.04077.x.

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: http://repository.cshl.edu/id/eprint/15539

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving