scholarly journals Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression

2021 ◽  
Vol 22 (22) ◽  
pp. 12344
Author(s):  
Jaemun Kim ◽  
Ji Yul Kim ◽  
Hyeonkyeong Kim ◽  
Eunsil Kim ◽  
Soonyong Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transfection Efficiency ◽  
Early Stage ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Early Period ◽  
Fluorescence Analysis ◽  
Stable Cell Line ◽  
Suspension Cells ◽  
Stage Of Development

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

scholarly journals CHO-S Antibody Titers >1 Gram/Liter Using Flow Electroporation-Mediated Transient Gene Expression followed by Rapid Migration to High-Yield Stable Cell Lines

2014 ◽  
Vol 20 (4) ◽  
pp. 545-551 ◽  
Author(s):  
Krista Steger ◽  
James Brady ◽  
Weili Wang ◽  
Meg Duskin ◽  
Karen Donato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Early Stage ◽  
Transient Gene Expression ◽  
Genetically Engineered ◽  
Expression Vectors ◽  
High Yield ◽  
Stable Cell Line ◽  
Cho Cell ◽  
Antibody Titers

In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

scholarly journals Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

2008 ◽  
Vol 24 (2) ◽  
pp. 458-465 ◽  
Author(s):  
S. Wulhfard ◽  
S. Tissot ◽  
S. Bouchet ◽  
J. Cevey ◽  
M. DeJesus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chinese Hamster Ovary ◽  
Mild Hypothermia ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Ovary Cells

scholarly journals Fetal Membrane Epigenetics

2020 ◽  
Vol 11 ◽  
Author(s):  
Tamas Zakar ◽  
Jonathan W. Paul
Keyword(s):  
Gene Expression ◽  
Early Stage ◽  
Expression Patterns ◽  
Normal Pregnancy ◽  
Clinical Samples ◽  
Fetal Membrane ◽  
Stage Of Development ◽  
Micro Rnas ◽  
Non Coding Rnas ◽  
Adverse Pregnancy

The characteristics of fetal membrane cells and their phenotypic adaptations to support pregnancy or promote parturition are defined by global patterns of gene expression controlled by chromatin structure. Heritable epigenetic chromatin modifications that include DNA methylation and covalent histone modifications establish chromatin regions permissive or exclusive of regulatory interactions defining the cell-specific scope and potential of gene activity. Non-coding RNAs acting at the transcriptional and post-transcriptional levels complement the system by robustly stabilizing gene expression patterns and contributing to ordered phenotype transitions. Here we review currently available information about epigenetic gene regulation in the amnion and the chorion laeve. In addition, we provide an overview of epigenetic phenomena in the decidua, which is the maternal tissue fused to the chorion membrane forming the anatomical and functional unit called choriodecidua. The relationship of gene expression with DNA (CpG) methylation, histone acetylation and methylation, micro RNAs, long non-coding RNAs and chromatin accessibility is discussed in the context of normal pregnancy, parturition and pregnancy complications. Data generated using clinical samples and cell culture models strongly suggests that epigenetic events are associated with the phenotypic transitions of fetal membrane cells during the establishment, maintenance and termination of pregnancy potentially driving and consolidating the changes as pregnancy progresses. Disease conditions and environmental factors may produce epigenetic footprints that indicate exposures and mediate adverse pregnancy outcomes. Although knowledge is expanding rapidly, fetal membrane epigenetics is still in an early stage of development necessitating further research to realize its remarkable basic and translational potential.

scholarly journals Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

2007 ◽  
Vol 29 (5) ◽  
pp. 703-711 ◽  
Author(s):  
Natalie Muller ◽  
Madiha Derouazi ◽  
Frédéric Van Tilborgh ◽  
Sarah Wulhfard ◽  
David L. Hacker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Cultivation Systems

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.

Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures

1985 ◽  
Vol 5 (5) ◽  
pp. 1188-1190
Author(s):  
T V Gopal
Keyword(s):  
Gene Expression ◽  
Transient Gene Expression ◽  
Stable Transformation ◽  
Suspension Cells ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Erythroleukemia Cells ◽  
Transfer Method ◽  
Gene Transfer Method ◽  
Mouse Myeloma

A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.

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