Transgenic, inducible RNAi in megakaryocytes and platelets in mice.
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.


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.
Molecular Medicine Division Chemical Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia.
M
M
Takiguchi
C
C
James
E C
EC
Josefsson
C L
CL
Carmichael
P K
PK
Premsrirut
S W
SW
Lowe
J R
JR
Hamilton
D C S
DC
Huang
B T
BT
Kile
R A
RA
Dickins
D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D034622:RNA Interference
D017931:DNA Primers; D049452:Green Fluorescent Proteins