Fig 1.
Different donor vectors were constructed.
Two independent promoters or one promoter plus IRES or 2A elements control light and heavy chain expression via single donor vectors. The UTRs are the areas, which transposase knows and cuts to transpose the donor vector into the host genome.
Table 1.
Sequences of primers used in the real-time PCR reactions.
Fig 2.
Flowcytometer analysis of created cell pools.
The pBLPCH vector contained GFP gene- used as the donor vector -, it was excited at 488 nm wavelength and—emitted at 532 nm. A, 24 hours post transfection. Transfection rates of all the three cell pools were 33–35%. B, After one month of selection with 10 μg/ml puromycin. Most of the cell populations had expressed GFP protein. C, six months post transfection and five months in non-selection medium the 1/2.5-pBLPCH cells retained GFP expression in more quantity than the other cell pools. Untransfected; The untransfected cells as the negative control, N-pBLPCH cells; created by pBLPCH only, 1/5-pBLPCH cells; created with 1:5 ratio of transposase/transposon (pBLPCH) vectors, and 1/2.5-pBLPCH cells; created with 1:2.5 ratio of transposase/transposon vectors.
Fig 3.
Generation and evaluation of dual ORF vector containing cells.
Three different cell pools were created, One by transfection of the pBLPCH vector only as a donor vector in the N-pBLPCH cells and the others by transfection of transposase and pBLPCH vectors with 1/5 and 1/2.5 ratios of helper to donor to produce 1/5-pBLPCH and 1/2.5-pBLPCH cells respectively. A, Western-blot analysis of expressed mAb in CHO cell pools supernatants in reduced and non-reduced forms. Goat anti-human HRP conjugated antibody was used as a detector. In the reduced form (left figure) Lane 1, N-pBLPCH cells, lane 2, 1/2.5-pBLPCH cells, lane3, 1/5-pBLPCH cells, Lane 4, pre-stained protein marker (10–170 kDa), lane 5; -untransfected cells supernatant (negative control), and lane 6; purified human IgG as a positive control, in the non-reduced western-blotting (right figure); lane 1,positive control, lane 2, protein marker, lane 3, N-pBLPCH, lane 4, 1/5-pBLPCH and lane 5, 1/2.5-pBLPCH. B, SDS-PAGE analysis. Protein-A purified mAbs were run in both reduced (lanes 1–4) and non-reduced (lanes 6–9) forms. Lanes 1 and 9; purified human IgG (positive controls), lanes 2 and 8; N-pBLPCH cells, lanes 3 and 7; 1/5-pBLPCH cells, lanes 4 and 6; 1/2.5-pBLPCH cells, and lane 5; protein marker (10–200 kDa). C, Cell pools antibody titers were measured by means of ELISA. Error bars indicate SD of triplicate measurements. ANOVA was used to detect statistically significant differences between the generated pools (P< 0.05).
Fig 4.
Expression evaluations of created pools by ELISA assay.
Expression levels of all the created pools evaluated by ELISA assay while quantitative real-time PCR was employed to determine gene copy numbers. Non-transposed cells had similar gene copy number although N-pBLIH cells had—low expression level. Transposition based cells had similar copy numbers as well, however, their expression levels were also more similar. Error bars indicate SD of triplicate measurements. ANOVA was used to detect statistically significant differences between the generated pools (P< 0.05).
Fig 5.
Western-blot analysis of single ORF donor vectors containing cell pools.
IRES and 2A elements were used to express light and heavy chains in bicistronic mRNAs. A, Western-blotting of IRES-containing cells in reduced and non-reduced forms. Lanes 1–3 are reduced and lanes 5–7 are non-reduced. Lane 1 and 7, purified IgG as a positive control, lane 2 and 6 purified mAb produced by N-pBLIH cells (due to their low expression profile western-blot had done on their purified product), lane 3and 5, the supernatant of 1/2.5-pBLIH cell pools, lane 4, pre-stained protein marker (10–170 kDa). B, Western-blot analysis of 2A harboring cells, left figure in reduced and right figure in non-reduced forms. In reduced picture, lane 1, positive control, lane 2, N-pBL2AH cells supernatant, lane 3, 1/2.5-pBL2AH cells supernatant, 25, 28 and 30 kDa light chains appeared in N-pBL2AH and 1/2.5-pBL2AH lanes respectively. Furin peptidase didn't work in the latter. In the non-reduced picture, lane 1, pre-stained protein marker (10–170 kDa), lane 2, N-pBL2AH and lane 3, 1/2.5-pBL2AH cells supernatants.
Fig 6.
mRNA levels in established pools were assessed by quantitative real-time PCR.
A, In dual promoter bearing cells both light and heavy chains, are expressed via an independent expression cassette. Transposition effect was assessed at the level of transcription of each chain and as it is demonstrated in the picture both chains expression rate increase in a similar way. HC to LC ratio analysis showed that HC in all dual promoter cells was produced in a greater amount. B, Light chain mRNA levels in single ORF mAb expressing cells were evaluated by real-time PCR using N-pBLPCH-LC mRNA as the control gene. Error bars indicate SD of triplicate measurements. ANOVA was used to detect statistically significant differences between the generated pools (P< 0.05).
Fig 7.
Stability study of dual promoter created pools.
Dual promoter cell pools were cultured about six months and their levels of expression were evaluated every other week by the aid of ELISA. As it is displayed in the picture transposition based cells maintained their superiority over the evaluation period but conventional created cells started losing their productivity after 10 weeks. N-pBLPCH cells; created by pBLPCH only, 1/5-pBLPCH cells; created with 1:5 ratio of transposase/transposon (pBLPCH) vectors, and 1/2.5-pBLPCH cells; created with 1:2.5 ratio of transposase/transposon vectors.
Fig 8.
mAb expression assessment in the clonal cell lines.
Dual promoter cell pools underwent clonal selection using limiting dilution method. A, In1/2.5-pBLPCH cells, 120 clones, in 1/5-pBLPCH cells, 130 clones, and in N-pBLPCH cells, 125 clones were recovered. Transposition based cells showed more productivity in comparison to conventional ones. B, More than 60% of clones in 1/2.5-pBLPCH cells had more than 1000μg/L expression while a similar percent of N-pBLPCH derivative cells had less than 100μg/L expression level. Around 21% of clones derived from 1/2.5-pBLPCH parental cells produced mAb more than 2500μg/L whereas two other groups had a comparable number of clones in this area and more than 10% of their clones expressed mAb in this level. C, Four clones of each pool were assessed for specific productivity and growth rates. As it is demonstrated, specific productivities of conventional clones were the lowest and transposition based clones were more productive. Clones growth rate were comparable in all evaluated clones. Error bars indicated SD of triplicate measurements.