Transgenic, Inducible RNAi in Megakaryocytes and Platelets in Mice

J Thromb Haemost. 2010 Dec;8(12):2751-6. doi: 10.1111/j.1538-7836.2010.04077.x.

Abstract

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.

MeSH terms

  • Animals
  • Base Sequence
  • Blood Platelets / metabolism*
  • Blotting, Western
  • Cells, Cultured
  • Cytomegalovirus / genetics
  • DNA Primers
  • Flow Cytometry
  • Green Fluorescent Proteins / genetics
  • Megakaryocytes / metabolism*
  • Mice
  • Mice, Transgenic
  • Promoter Regions, Genetic
  • RNA Interference*

Substances

  • DNA Primers
  • Green Fluorescent Proteins