Transgenic, inducible RNAi in megakaryocytes and platelets in mice

Takiguchi, M; James, C; Josefsson, EC; Carmichael, CL; Premsrirut, PK; Lowe, SW; Hamilton, JR; Huang, DCS; Kile, BT; Dickins, RA

Author(s): Takiguchi, M; James, C; Josefsson, EC; Carmichael, CL; Premsrirut, PK; Lowe, SW; Hamilton, JR; Huang, DCS; Kile, BT; Dickins, RA;
Details: Publication Year 2010-12,Volume 8,Issue #12,Page 2751-2756
Journal Title: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Publication Type: Journal Article
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.
Publisher: WILEY-BLACKWELL PUBLISHING, INC
Keywords: GENE-EXPRESSION; IN-VIVO; MAMMALIAN-CELLS; OVEREXPRESSION; DEATH
Publisher's Version: https://doi.org/10.1111/j.1538-7836.2010.04077.x
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2010-12-01 12:00:00