Aaron Lewis and Dr. Gregory Burton, Department of Chemistry and Biochemistry

RGS16 is an inhibitor of the chemokine receptor, CXCR4, and HIV uses CXCR4 to infect CD4+ T cells. CXCR4 is upregulated in germinal center (GC) T cells even though RGS16 is present in GC T cells. This reveals an interesting conundrum as to why CXCR4 is upregulated but is also inhibited by RGS16. My project entails cloning and expressing RGS16 to be further used in experiments that will determine why CXCR4 is upregulated and inhibited at the same time in germinal centers.

First, primers were designed for the amplification of RGS16 cDNA and a vector was chosen for cloning and expressing RGS16. The vector of choice was pCR 3.1. After the primers were designed, total mRNA was isolated from GC T cells, which were prepared from tonsils donated by Utah Valley Medical Center. The total mRNA was reverse transcribed followed by a polymerase chain reaction to amplify the RGS16 cDNA. Several trials of PCR were performed to optimize the reaction conditions. Primer and cDNA levels were altered from previous protocols to adjust for the small amount of RGS16 mRNA found in GC T cells. Upon amplifying RGS16, the cDNA was ligated into pCR 3.1 followed by transformation into one shot competent cells. The competent cells were used to copy the plasmid containing RGS16 so that a large quantity could be used to transfect eukaryotic cells. Jurkat cells are the choice of eukaryotic cells that will be used to express RGS16. Although RGS16 has not yet been isolated, the expressed protein will be isolated from the mammalian cells, and the isolated protein will be then used for future experiments that will explain why CXCR4 and RGS16 are co-expressed in germinal center T cells.