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The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells.

Kiseljak D, Rajendra Y, Manoli SS, Baldi L, Hacker DL, Wurm FM - BMC Proc (2011)

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédéral de Lausanne, CH-1015 Lausanne, Switzerland.

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Protein productivity in TGE has improved significantly over the past decade, reaching 300 mg/L and 1 g/L in CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively... In order to reduce the amount of plasmid DNA in TGE, we examined the possibility of partially replacing it with herring sperm DNA (non-coding “filler” DNA) in transfections of CHO and HEK cells... Transfections of 5 mL were performed in TubeSpin 50 bioreactors (TPP, Trasadingen, Switzerland) using 0.625 µg DNA/1x10 cells and 2.5 µg/1x10 cells of linear 25 kDa polyethyleneimine (PEI; Polysciences, Eppenheim, Germany). pA3 carrying the genes for a human IgG light and heavy chains was used for transfections... We tested the efficiency of herring sperm DNA as filler for TGE in CHO and HEK cells... The total amount of PEI was kept constant for all conditions... We observed that antibody titers increased when filler DNA was co-transfected with pA3 as compared to transfection with a reduced amount of pA3 alone (Fig. 1)... The results showed that the plasmid copy number decreased proportionally with the amount of pA3 transfected in the presence or absence of filler DNA in both CHO and HEK cells (data not shown)... Therefore, filler DNA did not influence the delivery of coding pDNA to transfected cells... However, when filler DNA was added to the complex, the release of pDNA from the complex was improved (data not shown)... Our data show that in TGE the amount of the coding vector could be reduced considerably by replacement of a significant proportion of pDNA with filler DNA (herring sperm DNA) without a major negative impact on recombinant protein productivity... However, filler DNA did not influence the delivery or stability of pDNA... The addition of filler DNA to the DNA-PEI complex, however, relaxed the complex in vitro... Based on these results, we speculate that the presence of filler DNA results in a more efficient intracellular release of the pDNA from the DNA-PEI complex and thus to improved transgene transcription.

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Effect of filler DNA on transient IgG production in A) CHO and B) HEK cells. IgG titers were measured on day 7 post-transfection by ELISA. The DNA amounts are presented in μg/106 cells.
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Figure 1: Effect of filler DNA on transient IgG production in A) CHO and B) HEK cells. IgG titers were measured on day 7 post-transfection by ELISA. The DNA amounts are presented in μg/106 cells.

Mentions: We tested the efficiency of herring sperm DNA as filler for TGE in CHO and HEK cells. We reduced the amount of pA3 to 17% or 33% of the optimum amount for each cell line and added filler DNA to 100%. The total amount of PEI was kept constant for all conditions. We observed that antibody titers increased when filler DNA was co-transfected with pA3 as compared to transfection with a reduced amount of pA3 alone (Fig. 1). These results showed that up to 83 % of the coding pDNA could be replaced by filler DNA with only a minimal negative impact on yield.


The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells.

Kiseljak D, Rajendra Y, Manoli SS, Baldi L, Hacker DL, Wurm FM - BMC Proc (2011)

Effect of filler DNA on transient IgG production in A) CHO and B) HEK cells. IgG titers were measured on day 7 post-transfection by ELISA. The DNA amounts are presented in μg/106 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285026&req=5

Figure 1: Effect of filler DNA on transient IgG production in A) CHO and B) HEK cells. IgG titers were measured on day 7 post-transfection by ELISA. The DNA amounts are presented in μg/106 cells.
Mentions: We tested the efficiency of herring sperm DNA as filler for TGE in CHO and HEK cells. We reduced the amount of pA3 to 17% or 33% of the optimum amount for each cell line and added filler DNA to 100%. The total amount of PEI was kept constant for all conditions. We observed that antibody titers increased when filler DNA was co-transfected with pA3 as compared to transfection with a reduced amount of pA3 alone (Fig. 1). These results showed that up to 83 % of the coding pDNA could be replaced by filler DNA with only a minimal negative impact on yield.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cellular Biotechnology, Faculty of Life Sciences, École Polytechnique Fédéral de Lausanne, CH-1015 Lausanne, Switzerland.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Protein productivity in TGE has improved significantly over the past decade, reaching 300 mg/L and 1 g/L in CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively... In order to reduce the amount of plasmid DNA in TGE, we examined the possibility of partially replacing it with herring sperm DNA (non-coding “filler” DNA) in transfections of CHO and HEK cells... Transfections of 5 mL were performed in TubeSpin 50 bioreactors (TPP, Trasadingen, Switzerland) using 0.625 µg DNA/1x10 cells and 2.5 µg/1x10 cells of linear 25 kDa polyethyleneimine (PEI; Polysciences, Eppenheim, Germany). pA3 carrying the genes for a human IgG light and heavy chains was used for transfections... We tested the efficiency of herring sperm DNA as filler for TGE in CHO and HEK cells... The total amount of PEI was kept constant for all conditions... We observed that antibody titers increased when filler DNA was co-transfected with pA3 as compared to transfection with a reduced amount of pA3 alone (Fig. 1)... The results showed that the plasmid copy number decreased proportionally with the amount of pA3 transfected in the presence or absence of filler DNA in both CHO and HEK cells (data not shown)... Therefore, filler DNA did not influence the delivery of coding pDNA to transfected cells... However, when filler DNA was added to the complex, the release of pDNA from the complex was improved (data not shown)... Our data show that in TGE the amount of the coding vector could be reduced considerably by replacement of a significant proportion of pDNA with filler DNA (herring sperm DNA) without a major negative impact on recombinant protein productivity... However, filler DNA did not influence the delivery or stability of pDNA... The addition of filler DNA to the DNA-PEI complex, however, relaxed the complex in vitro... Based on these results, we speculate that the presence of filler DNA results in a more efficient intracellular release of the pDNA from the DNA-PEI complex and thus to improved transgene transcription.

No MeSH data available.