Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

Grosjean, Frédéric; Bertschinger, Martin; Hacker, David L.; Wurm, Florian Μ.

doi:10.1007/s10529-006-9167-7

Cited by 16 publications

(11 citation statements)

References 16 publications

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“…Chemical‐based transfection methods are routinely used rather than viral transduction and physical methods such as electroporation, due to their ease of use with suspension cells and their ability to be easily scaled to large‐volume cultures. Common chemical delivery agents utilized for TGE include calcium phosphate,5–10 cationic lipids11–13 and cationic polymers. The cationic polymer polyethylenimine (PEI) is routinely used as a transfection reagent primarily because of its efficiency, cost‐effectiveness and ease of use in large‐scale transfections 3, 14, 15.…”

Section: Introductionmentioning

confidence: 99%

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Codamo

Hou

Hughes

et al. 2011

J of Chemical Tech & Biotech

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BACKGROUND: Transient gene expression (TGE) provides a rapid way to generate recombinant protein biologics for pre-clinical assessment. Human embryonic kidney (HEK293) cells have traditionally been used for TGE; however, there is demand from industry for efficient, high-producing TGE systems that utilize Chinese hamster ovary (CHO) cells. A polyethyleneimine (PEI) -based TGE process has been developed for CHO cells using an episomal expression system to generate enhanced recombinant protein titers.

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Section: Introductionmentioning

confidence: 99%

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Codamo

Hou

Hughes

et al. 2011

J of Chemical Tech & Biotech

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“…Many highly effective reagents are commercially available for gene delivery in small-scale transfection, but only a few are cost-effective enough for large-scale transfection applications. Calcium-mediated transfection has been well established (Batard et al, 2001;Chowdhury et al, 2003;Girard et al, 2001;Grosjean et al, 2002Grosjean et al, , 2006Jordan et al, 1998;Kofron and Laurencin, 2004;Lindell et al, 2004). Polyethylenimine (PEI), a cationic polymer, has also being used to deliver DNA into cells since it was first introduced (Boussif et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

High‐density transient gene expression in suspension‐adapted 293 EBNA1 cells

Sun

Hia

Goh

et al. 2007

Biotech & Bioengineering

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Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.

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“…Note: The addition of glycerol results in an osmotic shock and is thought to cause the release of the DNA from the precipitate ( Grosjean et al, 2006). …”

Section: Methodsmentioning

confidence: 99%

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines

Boehm¹,

Ott²

2017

BIO-PROTOCOL

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The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.

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Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

Cited by 16 publications

References 16 publications

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

High‐density transient gene expression in suspension‐adapted 293 EBNA1 cells

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines

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