Creating and Testing Tools for Investigating Gp93 Biology

Abstract

Mammalian glycoprotein 96 (gp96) and canopy 3 (CNPY3) are both required for chaperoning (folding) toll-like receptors (TLRs), infection recognition proteins, and integrins, cell adhesion molecules. Due to sequence similarity, just as gp96 and CNPY3 chaperone TLRs and integrins in mammals, we believe glycoprotein 93 (gp93) and canopy b (CNPYb) chaperone tolls and integrins in Drosophila melanogaster (fruit flies). To investigate this, we are constructing and testing multiple tools to study gp93 biology. First, toll and integrin genes are being cloned with tags to detect their expression in Drosophila S2* cells, as most do not have commercially available antibodies. Therefore, genes are being amplified from fly cDNA via polymerase chain reaction (PCR) and ligated into a cloning vector. Second, RNA interference (RNAi) constructs specific to chaperone genes must be made to knock down their expression in cells. Hence, multiple genes are being amplified via PCR and in vitro transcribed into RNAi. Lastly, integrin-specific antibodies are being tested via flow cytometry to confirm integrin expression in S2* cells. Thus far, multiple genes have been cloned, multiple RNAi constructs have been made, and we have verified integrin βPS expression in S2* cells. Collectively, these tools will enable us to study gp93 biology.