DGK Targeting via Transient and Stable Silencing

DGK-targeting was performed using the human DGKα (nt 1153-1173) and DGKζ (nt 2290-2310) validated sequences [35 (link), 36 (link), 71 (link)]. For transient silencing the sequences were transfected as dsRNA (Ambion) using oligofectamine (Invitrogen). A scramble dsRNA was used as control (Ambion). Effective DGK depletion was achieved at 72-96 h post-transfection. After 96h the expression was recovered. For stable silencing, 64 pb double strand oligonucleotides, encompassing the corresponding interfering 21 nt sequence and a hairpin structure, were cloned in the pSuperRetro vector (Oligoengine). As control the pSuperRetro construct harboring the homolog specific DGKζ mouse sequence was used. This control did not reduce the levels of human DGKα or ζ. SW480 cells were infected using standard protocols with the pSuperRetro-cloned sequence-containing retroviruses. Cells with the retrovirus insertion were selected by culturing in media with G418 (800 μg/ml) until the control, non-infected cells died (~1 week). The pools were checked for DGK silencing, maintained in media with G418 (500 μg/ml) and used in the long-term experiments.