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. 2005 Feb 8;33(3):846–856. doi: 10.1093/nar/gki223

Figure 4.

Figure 4

L1 siRNA can limit the activity of the RC-L1 clone, L1RP, in a transient retrotransposition assay. HCT116 or HeLa cells were transfected with pCEP4-L1RPneo alone or co-transfected with different siRNA. The retrotransposition activity was determined using a transient retrotransposition assay following G418 selection. The relative retrotransposition frequency of pCEP4-L1RPneo in the absence of siRNA was set at 100%. The negative control pCEP4-L1.3neo-JM111, an RC-L1 clone with two missense mutations in ORF1 rendering it inactive, showed no retrotransposition activity. (A) Results of the retrotransposition assay in HCT116 cells. Control siRNA used for transfection included 250 ng synthetic HIV Rev and 50 ng diced LacZ siRNA. Diced 5′-UTR siRNA (50 and 250 ng), diced ORF1 siRNA (25, 50 and 250 ng) and 250 ng 5′-UTR #749 synthetic siRNA were assayed separately. (B) Results of the retrotransposition assay in HeLa cells. Synthetic HIV Rev siRNA (250 ng) was included as a control. Diced 5′-UTR siRNA (50 and 250 ng) was also assayed. (C). The results of the retrotransposition assay achieved in HCT116 cells. The wild-type pCEP4-L1RPneo (or the negative control pCEP4-L1.3neo-JM111) was transfected alone or with the indicated amount of siRNA. G418 colonies were fixed and stained 14 days post-selection with 4% Giemsa for visualization.