RNA interference: A mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells
Section snippets
Materials and methods
Cells and cell culture. PANC1 pancreatic ductal adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco-BRL, Gaithersburg, MD). Cells were incubated in a humidified (37 °C, 5% CO2) incubator and passaged upon reaching 80% confluence.
Expression constructs and transfection. The pd2EGFP-1 vector was obtained from Clontech (Palo Alto, CA). The
Results and discussion
Overexpression of FLJ20174 fused to C-terminal FLAG sequence (DYKDDDDK) was confirmed in a transfectant clone (FLJ20174.1) derived from the well-characterized pancreatic ductal adenocarcinoma cell line PANC1 (Fig. 1). FLAG epitope migrated with an effective molecular weight of approximately 90 kDa, consistent with that predicted for FLJ20174. A chimeric protein comprising FLJ20174 fused to C-terminal green fluorescent protein (FLJ20174-GFP) localized predominantly to the cell membrane in
Acknowledgments
The authors gratefully acknowledge the technical assistance of Jan Rounds. This work was funded by The National Pancreas Foundation and departmental funding from the Department of Surgery, Brigham and Women’s Hospital.
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