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. 2016 Nov 29;5(11):e392. doi: 10.1038/mtna.2016.97

Figure 2.

Figure 2

Assessment of the U1-fix9 mediated rescue of hFIX splicing mutations in HEK293 stable clones. (a) Schematic representation of the snRNA U1-fix9 expression cassettes used. An AAV8 vector expressing the firefly luciferase under the control of the CMV promoter (AAV8-Luc) was used as control. In panels (b) and (d) RT-PCR and densitometry analyses on the total RNA of SChFIXex5-2C HEK293 clones transfected with the pU1-fix9 plasmid or transduced with the AAV8-U1-fix9. RT-PCR products were resolved by electrophoresis on 2% agarose gel. Negative controls are represented by untransfected or untransduced clones (−). The position of the primers used for the RT-PCR analyses is depicted. The quantification of exon 5 inclusion is performed as described above. Panels (c) and (e) show the quantification of hFIX protein levels in the media of SChFIXex5-2C HEK293 clones. Results are expressed as mean ±SD and derive from three independent experiments. Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. AAV, adeno-associated virus; HEK293, human embryonic kidney cells; SChFIX, splicing-competent human FIX transgene variants; SD, standard deviation; snRNA, small nuclear RNA; RT-PCR, reverse-transcription polymerase chain reaction; U1-fix9, ExSpeU1 fix9.