Figure 5: Promoters active in mammalian and insect cells.

(a) Structure of the tested dual promoters is shown schematically. In CMVP10, the baculoviral very late promoter p10 was inserted downstream of the CMV promoter. In CMVintP10, the p10 promoter was placed within an intron and is spliced out from the transcript of the CMV promoter. Grey arrow: transcription initiation of the CMV promoter; black arrow: transcription initiation of the p10 promoter. (b) HEK293, PAE and REF cells were infected with a MultiPrime baculovirus expressing EYFP-tubulin under the control of the above promoters and citrine under the control of the CMV promoter. The lysates of the cells were analysed by western blotting. (c) Quantification of blots shown in b. EYFP-tubulin/citrine ratio was used as a measure for promoter strength. Endogenous tubulin was used as loading control. The CMVintP10 promoter expresses at a similar level as the original CMV promoter, while the CMVP10 promoter expresses at a lower level in mammalian cells. (d,e) The same baculoviruses were used to infect insect (Sf21) cells. Citrine was expressed by a P10 driven expression cassette in the backbone of the baculovirus. Data shows mean value±s.d.; n=3; there is no significant difference (P>0.05) by comparing the two CMVP10 promoter variants using the Student’s t-test.