PU.1 is crucial for lymphomyeloid development, and its stage-specific expression is critical in preventing leukemic transformation.
However, such direct repression mechanisms are not the only way to down-regulate alternate lineage expression programs.
FACS analysis of bead uptake was performed on a FACSAria 3 sorter.
Bead-positive macrophages were FACS isolated from WT treated animals and stained with Wright-Giemsa stain, and were shown to generate phagocytotic vacuoles (data not shown).
In addition, 3000-5000 infected LSK cells were transplanted into lethally irradiated (9.5 Gy) syngenic recipients (SJL/C57/Bl6 CD45.1) with 5 × 10 In brief, for in vitro studies peritoneal washout cells were incubated in IMDM for 1 hour and then 20 μL/m L of beads were added for 90 minutes.
In vivo phagocytosis was measured by instilling a diluted bead solution IP 18 hours before collection of peritoneal washout.
I restriction site) ttcctcgagccagcatgtttaatgattgctt. The correct insertion of the pre-mi R was confirmed by sequencing.
The mi R expression plasmids were then cotransfected using calcium-phosphate precipitates (Invitrogen) with gag/pol and eco-env plasmids into HEK293T cells, and virus-containing supernatant was harvested 3 and 4 days later, filtered, and stored at −80°C until use.
In agreement with this observation, disruption of Transcription factors play a decisive role in determining lineage fate from HSCs.
To test for such negative regulatory events, we searched for PU.1-controlled micro RNAs (mi Rs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model.