The ligands for many NK cell receptors remain unknown, thus identification of these ligands has become a major goal for understanding the molecular basis of regulation of NK cell response to cancer invasion.
Receptor-Ig(Fc) is a fusion of extracellular portion of an NK/T cell receptor and human IgG(Fc). These chimeric proteins recently became a common and a very effective tool used in contemporary biomedical research. The unique properties, given by fusion of antibody heavy chain with a protein domain of interest, make this construct useful for generating soluble receptor constructs that can be used to identify ligands in FACS, ELISA or Fluorescent Imaging based assays.
We established a unique fully automated protocol and the necessary controls to perform a whole genome siRNA screen to identify novel NK cell receptors ligands. We hypothesized that siRNAs targeting the ligand or proteins important for surface expression of the ligand would significantly diminish Receptor-Ig cell surface staining. Therefore subsequent image analysis of certain siRNA-transfected cells will allow us to determine screens Hits - lack of staining. Exploiting this original approach we successfully identified number of potential ligands for NK cell receptors.
