Scalable Transient Gene Expression in Adherent Mammalian Cells Using Polyethylenimine

2013 ◽  
pp. 29-34
Author(s):  
Lukas Fliedl ◽  
Christian Kaisermayer
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Transient Gene Expression ◽  
Adherent Mammalian Cells

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency

1982 ◽  
Vol 2 (9) ◽  
pp. 1145-1154
Author(s):  
Y M Shen ◽  
R R Hirschhorn ◽  
W E Mercer ◽  
E Surmacz ◽  
Y Tsutsui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Thymidine Kinase ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Transient Gene Expression ◽  
Simian Virus 40 ◽  
Temperature Sensitive ◽  
Enzymatic Function ◽  
Very High

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

Large Scale Transient Gene Expression in Mammalian Cells

2006 ◽  
pp. 113-116
Author(s):  
E. -J. Schlaeger ◽  
K. Christensen ◽  
G. Schmid ◽  
N. Schaub ◽  
B. Wipf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Large Scale ◽  
Transient Gene Expression ◽  
Expression In Mammalian Cells

Production and characterization of HIV-1 virus-like particles using transient gene expression in mammalian cells

2014 ◽  
Vol 31 ◽  
pp. S42
Author(s):  
Sonia Gutiérrez-Granados ◽  
Laura Cervera ◽  
Segura Maria de las Mercedes ◽  
Francesc Gòdia
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Transient Gene Expression ◽  
Virus Like Particles ◽  
Expression In Mammalian Cells ◽  
Hiv 1

scholarly journals Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

2020 ◽  
Author(s):  
Matthew Stuible ◽  
Christian Gervais ◽  
Simon Lord-Dufour ◽  
Sylvie Perret ◽  
Denis L’Abbe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Transient Gene Expression ◽  
Full Length ◽  
Spike Protein ◽  
Hek293 Cells ◽  
High Yield ◽  
Diagnostic Tools ◽  
Purification Method

ABSTRACTRecombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

scholarly journals Characterizing the Non-Normal Distribution of Flow Cytometry Measurements from Transiently Expressed Constructs in Mammalian Cells

2016 ◽  
Author(s):  
Peter F. McLean ◽  
Christina D. Smolke ◽  
Marc Salit
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Normal Distribution ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Transient Gene Expression ◽  
Single Copy ◽  
Asymmetric Distribution ◽  
Experimental Conditions ◽  
Fluorescent Reporter

AbstractIn mammalian cells, transient gene expression (TGE) is a rapid, minimal-investment alternative to single-copy integrations for testing of transgenic constructs. However, transient gene expression, as measured by flow cytometry with a fluorescent reporter, typically displays a broad, asymmetric distribution with a left-tail that is convolved with background signal. Common approaches for deriving a summary statistic for transiently expressed gene products impose a normal distribution on gated or ungated data. Summary statistics derived from these models are heavily biased by experimental conditions and instrument settings that are difficult to replicate and insufficient to accurately describe the underlying data. Here, we present a convolved gamma distribution as a superior model for TGE datasets. The 4-6 parameters of this model are sufficient to accurately describe the entire, ungated distribution of transiently transfected HEK cells expressing monomeric fluorescent proteins, that operates consistently across a range of transfection conditions and instrument settings. Based on these observations, a convolved gamma model of TGE distributions has the potential to significantly improve the accuracy and reproducibility of genetic device characterization in mammalian cells.

Transient Gene Expression‐Based Protein Production in Recombinant Mammalian Cells

2019 ◽  
pp. 49-72 ◽  
Author(s):  
Joo‐Hyoung Lee ◽  
Henning G. Hansen ◽  
Sun‐Hye Park ◽  
Jong‐Ho Park ◽  
Yeon‐Gu Kim
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Protein Production ◽  
Transient Gene Expression
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