In Vitro Analysis of Psoriasis Linked CARMA2sh Gene in Keratinocytes and Mouse Embryonic Stem Cells towards Developing a Murine Transgenic Model
Abstract
Psoriasis is a debilitating skin disease affecting approximately 23% of
human population. Genome-wide association studies have identified
more than 40 susceptibility loci for psoriasis. CARMA2 proteins play a
major role in regulating activation of NF-kB, which controls inflammatory
response, cell survival, and proliferation. Missense mutations in the
CARMA2 gene have been shown to dominantly transmit the psoriatic
trait with high penetrance. It has been identified that CARMA2sh induces
activation of NF-κB and this requires the function of another CARD-containing
protein, BCL10 and TRAF2. Recently a novel CARMA Inhibitory
Kinase (CIK) which inhibits the ability to induce NF-kB was identified;
however these molecules are not tested in Human Primary Keratinocytes
(HEK). We attempted to study the function of CIK and its associated molecules
by in vitro and in vivo models. Our objective is to investigate the
activity of CIK on HEK cells expressing wild and mutant CARMA2sh
forms. Standard culturing method was used to maintain the normal human
epidermal keratinocytes (HEK) cells. Active or inactive forms of CIK
and CARMA2sh expression vectors were constructed. Transformation in
DH5α E.coli cells was followed by transfection of respective constructs in
HEK cells. Expression levels of target gene were analyzed by real-time
PCR, immune blot, and immunohistochemistry. To confirm the target
gene in mouse embryonic stem (ES) cells, generation of CARMA2sh
mutant associated with psoriasis (Gly117Ser and Glu 138Ala) were
created by site-directed mutagenesis and generating transgene via
homologous recombination method. The gene targeting constructs
electroporated into ES cells. Targeted ES cell clones confirmed by PCR
and southern blot. Positive clones were microinjected into blastocysts and
implanted into 10-15 pseudo-pregnant females. It has been observed that
with in vitro analysis, active form of CIK significantly reduced the expression
of mutant CARMA2sh gene in HEK cells. The gene targeted
positive clones were successfully electroporated and expressed in mouse
ES cells. Future studies will investigate the effect of CARMAsh RNA mediated
knockdown CIK on the activation of NF-kB targeted genes and
signal transduction pathways that control cell death and proliferation.
DOI/handle
http://hdl.handle.net/10576/5723Collections
- Laboratory Animal Research Center (Research) [97 items ]