Figure 1.
Schematic drawings of plasmids used to generate the influenza replicon based expression system.
pTripolis and pBi-NP generate the influenza replicon (mRNA/protein and vRNA) whereas pMono plasmids with various genes of interest or reporter genes drive transcription of vRNA.
Figure 2.
Schemata of bidirectional and monodirectional transcription cassettes.
Bidirectional cassettes (A) were used for generation of the influenza replicon. Monodirectional cassettes (B) were used to generate vRNA of reporters or genes of interest.
Figure 3.
Transfection efficiency time course.
HEK-293 cells either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu Replicon) were monitored over a period of 101 hours by FACS analysis. Data represents arithmetic mean values and standard deviation.
Figure 4.
HEK-293 cells were either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu Replicon). Pictures were taken 56 and 98 hours post transfection. Non-transfected cells were used as negative control (Neg. Control).
Table 1.
Plasmids and plasmid amounts used for various experiments.
Figure 5.
FACS analysis of cells transfected with either pEGFP-N1 (CMV) or the influenza replicon system and pMono-EGFP (Flu Replicon) 77 hours post transfection.
Cells transfected with the replicon system show two very distinct populations in the forward light scatter (EGFP), one completely negative, the other highly positive. pEGFP-N1 driven EGFP expressions shows a broad distribution spanning from completely negative to moderate and high expressing cells.
Figure 6.
Time course of firefly luciferase expression.
HEK-293 cells either tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Flu Replicon) were monitored over a period of 101 hours by luciferase assay. Data represents arithmetic mean values and standard deviation.
Figure 7.
Comparison of firefly luciferase expression driven by CMV, SV40 or by the influenza replicon.
HEK-293 cells were either tranfected with pcDNA-luc (CMV), pGL3-control (SV40) or with the influenza replicon and pMono-lucNS (Influenza Replicon) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.
Figure 8.
Activity of the influenza replicon in a human, a canine and a rodent cell line.
HEK -293, MDCK or CHO-K1 cells were either tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Influenza Replicon). The HEK-293 negative control has been transfected with pMono-lucNS and pBi-NP only. Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. In case of MDCK and CHO-K1, untransfected cells have been used as negative controls and can therefore not be normalized, absolute values can be found in Results section. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Data represents arithmetic mean values and standard deviation.
Figure 9.
Temperature dependence of the influenza replicon.
HEK-293 cells were either tranfected with pcDNA-luc (CMV), pGL3-control (SV40) or with the influenza replicon and pMono-lucNS (Flu Replicon) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Four hours post transfection cells were transferred to 27°C or 40°C or remained at 37°C. Cells were harvested 48 hours post transfection and were subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.
Figure 10.
Influence of different NCRs on the expression level
. HEK-293 cells were tranfected with the influenza replicon and either pMono-lucNS (NCR NS) or pMono-lucM (NCR M) or pMono-lucPB1 (NCR PB1) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.
Figure 11.
Expression of a the monoclonal anti-gp41 antibody 3D6.
HEK-293 cells were either tranfected with pRC-LC and pRC-HC (CMV HC∶LC = 1∶1, CMV HC∶LC = 10∶1) or with the influenza replicon and pMono-LC and pMono-HC (Flu Replicon HC∶LC = 1∶1, Flu Replicon HC∶LC = 10∶1). Heavy chain to light chain ratios of 1∶1 and 10∶1 were tested. Antibody concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.
Figure 12.
HEK-293 cells were either tranfected with pRC-LC (CMV) or with the influenza replicon and pMono-LC (Flu Replicon). Light chain concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.