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Figure 7 | BMC Molecular Biology

Figure 7

From: Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns

Figure 7

αDENV-GrpI -FL constructs effectively target and suppress DENV-2 in mosquito cells. A) Confirmation of representative αDENV-GrpI trans-splicing activities. Ae. Albopictus C6/36 cells were transiently transfected with trans-splicing αDENV-GrpI bicistronic vector constructs. Resulting RNAs were analyzed by RT-PCR in the presence (+Rt) or absence (-Rt) of reverse transcriptase to insure observed amplified products were derived from RNA. A PCR amplification product derived from a constructed spliced sequence control (Methods) is provided as a size standard for each gel (DNA+Ctrl). Δ9 and Δ96 refer to Pabc5 deletion mutations located in the trans-splicing domain of the group I intron. These deletions are designed to knock out function providing an adequate negative control [52]. Arrows indicate the predicted size of the principle splice products resulting from intron activity. The identity of spliced product was confirmed by sequencing. B) C6/36 cells were transformed with trans-splicing αDENV-GrpI bicistronic vector constructs and maintained under 10 mg/ml hygromycin selection for more than 30 doublings. Transformed cells were washed twice in serum free media at 15 hours post plating, infected with DENV-2 NGC (MOI = 0.1). RNAs were analyzed by RT-PCR as described in A). Arrows indicate the predicted size of the principle splice products. C) Ae. albopictus C6/36 cells were transformed with group I intron vector constructs and maintained under hygromycin selection. Transformed cells were washed three times and challenged with DENV 2-NGC (MOI 0.01). Infections were allowed to proceed for 4 days, supernatants were collected, and viral titers were determined by TCID50-IFA as described in Methods.

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