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Role of non‐specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK‐293E cells
Author(s) -
Rajendra Yashas,
Kiseljak Divor,
Manoli Sagar,
Baldi Lucia,
Hacker David L.,
Wurm Florian M.
Publication year - 2012
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24494
Subject(s) - chinese hamster ovary cell , transfection , dna , transgene , recombinant dna , hek 293 cells , microbiology and biotechnology , exogenous dna , gene , plasmid , coding region , biology , gene expression , cell culture , chemistry , biochemistry , genetics
Abstract Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non‐specific (filler) DNA to partially replace the transgene‐bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK‐293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady‐state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA–polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA–PEI complexes, leading to a higher percentage of transfected cells. The co‐assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA. Biotechnol. Bioeng. 2012;109: 2271–2278. © 2012 Wiley Periodicals, Inc.