Fig 1.

(A) (Top) Strategy of subcloning U6-shRNA cassette from TRC shRNA plasmid into L1 retrotransposition vector. Blue and green lines represent the adaptor sequences for LIC cloning. (Bottom) Sequence of LIC adaptor sequence used in this study. The adaptor includes sequences highlighted with blue and green and a BstZ17I site (underlined). Residues in red represent the boundaries of overhangs after T4 polymerase treatment. (B) Relative L1 retrotransposition efficiencies, with and without U6-shRNA cassette, for native human L1.3 and synthetic human L1 (ORFeus-Hs). The absolute value of L1 activity from the vector without the U6-shRNA cassette was 8% and was arbitrarily set at 1 for comparison. Six independent experiments were done, and standard errors are shown. (C) (Top) Subcloned anti-eGFP shRNA efficiently knocked down eGFP expression in HeLa cells. (Bottom) Subcloned anti-eGFP shRNA efficiently knocked down eGFP expression in HEK293T cells. Cells were cotransfected with pCEP-puro GFP (0.5 μg) and pLD108-shRNA (1.0 μg) plasmids, and eGFP expression was monitored at different time points until 96 h posttransfection. The numbers at the bottom represent relative fluorescence signals of the bottom row (with anti-eGFP shRNA) compared to the top row (with scrambled shRNA).