In Vitro Analysis of Psoriasis Linked CARMA2sh Gene in Keratinocytes and Mouse Embryonic Stem Cells towards Developing a Murine Transgenic Model
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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.
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