Abstract
RNA mediated gene silencing, RNA interference (RNAi), has recently been
identified in mammalians. This thesis describes the utilization and
development of RNAi based tools for signal transduction research. The
effects of Bruton s tyrosine kinase (Btk) down-regulation with RNAi have
been studied with gene expression profiling in hematopoietic cells.
Btk is indispensable for the B cell development. The mutation in the Btk
gene causes primary immunodeficiency disease X-linked agammaglobulinemia
(XLA) in humans and X-linked immunodeficiency (Xid) in mice. Less is
known about the Btk s role in other hematopoietic cells.
This work addresses Btk s role in monocytes, macrophages and mast cells.
In paper I, Btk s down-regulation with short interfering RNA (siRNA)
caused decreased histamine secretion in the RBL-2H3 mast cell line.
Moreover, the concept of using three siRNA against same target gene for
enhanced down regulation was introduced for the first time. In paper II,
U937 cell line expressing short hairpin RNA (shRNA) against Btk was
created. The outcome of stable Btk down-regulation was analyzed with gene
expression profiling. Btk was found to regulate 58 transcripts in
macrophages (PMA stimulated U937 cells) compared to 11 in monocytes. The
analysis suggests Btk s involvement in macrophage effector functions. In
paper III, an inducible shRNA vector system was developed to overcome the
shortcomings of constitutively expressed shRNAs. The system uses Cre
recombinase-mediated site-specific recombination, H1 polymerase III
promoter-driven expression and lentiviral delivery. The advantages of
pLIND (LentiINDucible) vector system are, no basal shRNA expression in
the inactive state, EGFP selection marker and lentiviral transduction
enabling gene transfer into primary and other difficult-to-transfect
cells. In paper IV, the potential Btk dependency of bone marrow-derived
mast cells (BMMC) was addressed. Btk deficient BMMC were shown to grow
slower compared to parental cells. This was found to be due to G2/M
arrest. Moreover, it was found that Btk negatively regulates expression
of novel G protein coupled receptor GPR177. This negative regulation was
also found in B cells. Another affected transcript was up-regulation of
melanoma antigen (Mela), which is of interest owing to Btk s role as a
putative tumor suppressor gene.