Conditional overexpression of transgenes in megakaryocytes and platelets in vivo.
Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic beta-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by beta-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.
Nguyen
Hao G
HG
Boston University School of Medicine, Department of Biochemistry, 715 Albany St, K225, Boston, MA 02118, USA.
Yu
Guangyao
G
Makitalo
Maria
M
Yang
Dan
D
Xie
Hou-Xiang
HX
Jones
Matthew R
MR
Ravid
Katya
K
D000818:Animals; D064107:Aurora Kinase B; D064127:Aurora Kinases; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004318:Doxycycline / Q000008:administration & dosage; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D010641:Phenotype; D010976:Platelet Count; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013752:Tetracycline / Q000008:administration & dosage; D019076:Transgenes / Q000187:drug effects / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; C576924:Aurkb protein, mouse; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D001616:beta-Galactosidase; D013752:Tetracycline; D004318:Doxycycline