Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

Pradhan, Ketaki; Gadgil, Mugdha

doi:10.1007/s10616-012-9435-4

Cited by 10 publications

(12 citation statements)

References 29 publications

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“…Plasmid DNA (125–500 ng) and, where indicated, siRNA oligonucleotides (final concentration of 50 nM) were added to 100 μl of jetPRIME buffer. In addition, 125 ng of salmon sperm DNA (UltraPure Salmon Sperm DNA Solution, Thermo Fisher Scientific) or 40 nM of the 21‐mer oligonucleotide CGUACGCGGAAUACUUCGAdTdT (Elbashir et al , ) was added to the plasmid DNA per transfection, serving as carrier to increase the transfection efficiency as previously described (Pradhan & Gadgil, ). 2 μl of jetPRIME was added, followed by 10‐min incubation, before the mixture was added to the cells for 24 h before fixation.…”

Section: Methodsmentioning

confidence: 99%

HP 1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

et al. 2018

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The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP‐B DNA‐binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore–microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1‐CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G2. HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1‐independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G2 before Aurora B‐mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.

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Section: Methodsmentioning

confidence: 99%

HP 1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

et al. 2018

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“…Transfection efficiency has been enhanced by various strategies based on reporter genes, or by optimization or addition of conditions or factors such as transfection reagents, 5 DNA topology of plasmid, 11 cell cycle distribution of host cells, 12 and non-encoding carrier DNA. 13 The Epstein-Barr virus nuclear antigen-1 (EBNA-1) gene was shown to accelerate gene delivery and promote transcription. Transfection efficiency and protein expression in electroporation were enhanced when this gene was inserted in the expression plasmid.…”

Section: Discussionmentioning

confidence: 99%

A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye

et al. 2016

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Enhanced transfection efficiency of transient gene expression (TGE) and electroporation is a useful approach for improvement of recombinant therapeutic proteins in mammalian cells. A novel method is described here in which CHO cells expressing recombinant FVII (rFVII) were labeled with fluorescent dye and analyzed by confocal microscopy. Cells with or without rFVII encoding gene were detectable by flow cytometry. Thus, we were able to distinguish positive cells (with rFVII encoding gene) and quantify their percentages. We evaluated the effects of varying electroporation conditions (voltage, number of repetitions, plasmid amount, carrier DNA) in order to optimize transfection efficiency. The highest transfection efficiency achieved was ∼86%. The method described here allows rapid evaluation of transfection efficiency without co-expression of reporter genes. In combination with appropriate antibodies, the method can be extended to evaluation of transfection efficiency in cells expressing other recombinant proteins.

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“…Plasmid DNA [50] and salmon sperm DNA [51] were used as carrier DNA for chemical transfection. In our conditions, however, the addition of salmon sperm DNA completely inhibited the enhancer-supported transfection (data not shown).…”

Section: Discussionmentioning

confidence: 99%

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

et al. 2015

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Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

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Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

Cited by 10 publications

References 29 publications

HP 1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

HP 1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

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