Strategies for Efficient Transfection of CHO-Cells with Plasmid DNA

2011 ◽  
pp. 213-226 ◽  
Author(s):  
Renate Kunert ◽  
Karola Vorauer-Uhl
Keyword(s):  
Plasmid Dna ◽  
Cho Cells

scholarly journals Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
James D. Budge ◽  
Robert J. Young ◽  
Christopher Mark Smales
Keyword(s):  
Recombinant Protein ◽  
Plasmid Dna ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Nuclear Antigen ◽  
Stable Cell Line ◽  
Nuclear Pore

Transient gene expression (TGE) in mammalian cells is a method of rapidly generating recombinant protein material for initial characterisation studies that does not require time-consuming processes associated with stable cell line construction. High TGE yields are heavily dependent on efficient delivery of plasmid DNA across both the plasma and nuclear membranes. Here, we harness the protein nucleoside diphosphate kinase (NDPK-A) that contains a nuclear localisation signal (NLS) to enhance DNA delivery into the nucleus of CHO cells. We show that co-expression of NDPK-A during transient expression results in improved transfection efficiency in CHO cells, presumably due to enhanced transportation of plasmid DNA into the nucleus via the nuclear pore complex. Furthermore, introduction of the Epstein Barr Nuclear Antigen-1 (EBNA-1), a protein that is capable of inducing extrachromosomal maintenance, when coupled with complementary oriP elements on a transient plasmid, was utilised to reduce the effect of plasmid dilution. Whilst there was attenuated growth upon introduction of the EBNA-1 system into CHO cells, when both NDPK-A nuclear import and EBNA-1 mediated technologies were employed together this resulted in enhanced transient recombinant protein yields superior to those generated using either approach independently, including when expressing the complex SARS-CoV-2 spike (S) glycoprotein.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Polymeric Delivery of Proteins and Plasmid DNA for Tissue Engineering and Gene Therapy

2001 ◽  
Vol 11 (1-3) ◽  
pp. 12 ◽  
Author(s):  
Thomas P. Richardson ◽  
William L. Murphy ◽  
David J. Mooney
Keyword(s):  
Tissue Engineering ◽  
Gene Therapy ◽  
Plasmid Dna ◽  
Polymeric Delivery

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation

Evaluation of biocompatibility and efficiency of plasmid DNA delivery by gene-activated hydrogels in vitro

2019 ◽  
Vol XIV (2) ◽  
Author(s):  
I.Y. Bozo ◽  
A.A. Titova ◽  
M.N. Zhuravleva ◽  
A.I. Bilyalov ◽  
M.O. Mavlikeev ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Delivery

Detection of Rhizoctonia solani AG-2-2 IV, the Causal Agent of Large Patch of Zoysiagrass, Using Plasmid DNA as a Probe.

1998 ◽  
Vol 64 (5) ◽  
pp. 451-457 ◽  
Author(s):  
Susumu TAKAMATSU ◽  
Manami NAKANO ◽  
Hideyuki YOKOTA ◽  
Hitoshi KUNOH
Keyword(s):  
Rhizoctonia Solani ◽  
Plasmid Dna ◽  
Causal Agent ◽  
Large Patch
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