a, Luciferase assay to determine the efficiency of target suppression by the pHippy vector. Hairpin siRNA expression vectors were generated against PGL3 luciferase with either the U6 (U6GPL31ucHP) or H1 (HlPGL31ucHP) promoters. For comparison, pHippy siRNA vectors were generated against PGL3 luciferase (pHippyPGL31uc) or against EGFP (pHippyEGFP). 293T cells were transfected with the constructs shown and assayed for luciferase activity 24 hours later. The absolute levels of PGL3 luciferase were ~100,000 relative light units (RLUs) which was set at 100%. All experiments were normalized for transfection efficiency with an expression vector for Renilla luciferase. The average normalized PGL3 luciferase levels and standard deviation are shown for 3 experiments. b, EGFP fluorescent assay to monitor the suppression of protein expression by pHippyEGFP. 293T cells were co-transfected with empty pHippy or pHippyEGFP and expression vectors for DsRED and EGFP. Confocal image of EGFP and EGFP merged with DsRED are shown. One representative experiment is shown.
Kaykas and Moon BMC Cell Biology 2004 5:16 doi:10.1186/1471-2121-5-16