Gromadzka, A.M., Steckelberg, A.-L., Singh, K.K, Hofmann, K. and Gehring, N.H.
Notes: NanoLuc® and Firefly luciferase reporters (pCMV128-NanoLuc and pCI-FireflyLuc) were co-transfected into HeLa cells to provide complementary measures of specific mRNA splicing and export activity. In the presence of splicing activity, firefly luciferase pre-mRNA is spliced in the nucleus, exported and translated resulting in firefly luciferase activity. In contrast, the open reading frame of the Nanoluc luciferase in pCMV128 NanoLuc® was placed between two weak splice sites. If the pre-mRNA is spliced, the NanoLuc coding sequence is removed and no luciferase can be expressed. The NanoLuc® reporter can only be translated when the unspliced pre-mRNA is transported to the cytoplasm. pCMV128 NanoLuc® was generated by inserting the NanoLuc® sequence into the NotI and BamHI sites of pCMV128, and then used for export assays. Cells were harvested 72 h post-transfection using 300 μl Passive Lysis Buffer. Lysates were analyzed using the NanoGlo® Dual-Luciferase® Reporter Assay. (4744)