Pshuttle cmv

Human breast adenocarcinoma MCF-7 and T-47D cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and both cell lines were maintained in DMEM (Life Technologies, Grand Island, NY) plus 10 mM HEPES (Mediatech, Herndon, VA) and 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO). The yCD coding sequence was fused to the C-terminal end of human SSTR2 via a seven amino acid linker in an overlap extension polymerase chain reaction. The resulting product was cloned into pShuttle-CMV (Agilent Technologies, Santa Clara, CA) via XhoI and EcoRV, sites for which were incorporated into the construct via two primers for the PCR, and the resulting plasmid was pS-SSTR2-yCD. The clone was sequence verified to show the absence of mutations. Both the SSTR2 and yCD portions of the fusion were then validated in functional assays in transiently transfected MCF-7 and T-47D cells. Following the validation, pS-SSTR2-yCD was used to produce recombinant adenoviral plasmid, which was then transfected into AD-293 cells (ATCC) for the production of crude SSTR2-yCD adenovirus, which was sent to Q-Biogene (Montreal, Canada) for the production of purified AdSSTR2-yCD. AdSSTR2 was produced as previously described.^{21 (link)}