A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse, Caroline; Burchell, Joy; Gschmeissner, Stephen; Brockhausen, Inka; Lloyd, Kenneth O.; Taylor-Papadimitriou, Joyce

For production of an amphotrophic ␣ 2,3 sialyltransferase-expressing retrovirus, the HindIII/XbaI fragment was excised from pBluescript, blunt ended with Klenow, and subcloned into the SnaBI site of the pBabe puro vector. The amphitrophic packaging cell line AM12 was transfected with this construct using calcium phosphate-mediated transfection. Briefly, AM12 cells were grown to 70% confluency in 10-cm tissue-culture dishes and refed with fresh medium 1 h before transfection. 15 g of pBabepuro3-STMYC DNA and 25 g of carrier salmon sperm DNA were coprecipitated with calcium phosphate at pH 7. After 6 h at 37 Њ C, dishes were rinsed five times with serum-free medium and then refed with fresh medium. 48 h after transfection, cells were split 1:10 into growth medium containing 2 g/ml puromycin for selection. Medium was changed every 3-4 d for 4 wk until selection was complete. The ␣ 2,3 sialyltransferase-retrovirus producer cell line was grown to 70% confluency, and the spent medium was replaced with half the volume of fresh medium. 3 d later, the virus-containing medium was removed, filtered, quick frozen in dry ice, and stored at Ϫ 70 Њ C.