PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

Xie, Qiuling; Xinyong, Guo; Chen, Xianjin; Wang, Yayu

doi:10.1007/s10616-012-9483-9

Cited by 39 publications

(24 citation statements)

References 28 publications

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“…From the above data, it is clear that the CpTi discs coated with PEI/ pEGFP nanoplexes prepared at N/P ratios of 15 and 20 demonstrated high cytotoxicity which is probably due to the increased toxicity of free uncomplexed PEI. (33) These results are consistent with previous studies which reported that increasing the N/P ratio of nanoplexes increases cytotoxicity. (34)…”

Section: Resultssupporting

confidence: 93%

Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Atluri¹,

Lee²,

Seabold³

et al. 2017

Int J Oral Maxillofac Implants

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Commercially pure titanium (CpTi) and its alloys possess favorable mechanical and biological properties for use as implants in orthopedics and dentistry. However, failures in osseointegration still exist and are common in select individuals with risk factors such as smoking. Therefore, in this study, a proposal was made to enhance the potential of CpTi discs for osseointegration by coating their surfaces with nanoplexes comprising polyethyleneimine (PEI) and plasmid DNA encoding bone morphogenetic protein-2 (pBMP-2). The nanoplexes were characterized for size and surface charge at a range of N/P ratios. CpTi discs were surface characterized for morphology and composition before and after nanoplex coating using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRD). The cytotoxicity and transfection ability of CpTi discs coated with nanoplexes of varying N/P ratios in human bone marrow derived mesenchymal stem cells (BMSCs) was measured via MTS assays and flow cytometry, respectively. The CpTi discs coated with nanoplexes prepared at an N/P ratio of 10 (N/P-10) were considered optimal, resulting in 75% cell viability and 14% transfection efficiency. ELISA results demonstrated a significant enhancement in BMP-2 protein secretion by BMSCs 7 days post-treatment with CpTi discs coated with PEI/pBMP-2 nanoplexes (N/P-10), compared to the controls. Real time PCR data demonstrated that the BMSCs treated with PEI/pBMP-2 nanoplex coated CpTi discs resulted in an enhancement of runx-2, alkaline phosphatase and osteocalcin gene expressions on day 7, post-treatment. In addition, these BMSCs demonstrated enhanced calcium deposition on day 30 post-treatment as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays. Thus, from all the above data it can be concluded that PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs have the potential to induce osteogenesis and enhance osseointegration.

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Section: Resultssupporting

confidence: 93%

Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Atluri¹,

Lee²,

Seabold³

et al. 2017

Int J Oral Maxillofac Implants

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“…2). It is known that PEI can be cytotoxic to cells (Boeckle et al, 2004) and a similar initial drop in viability has been reported for other PEI-based transfections (Backliwal et al, 2008;Codamo et al, 2011;Mozley et al, 2014;Xie et al, 2013). Upon the addition of feed (Tryptone N1) 24 h posttransfection, the cells began to grow and viability recovered, an effect in keeping with reports of others on cell growth and recombinant protein production (Pham et al, 2005).…”

Section: Differences In Timp Protein Production Are Not Due To Effectsupporting

confidence: 82%

Use of a protein engineering strategy to overcome limitations in the production of “Difficult to Express” recombinant proteins

Hussain

Fisher

Abbott

et al. 2017

Biotech & Bioengineering

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Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.

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“…Transfection and expression are influenced by different PEI to DNA ratios as it affects the diameter and stability of the DNA:PEI complex. 39 By testing a variety of N:P ratios and DNA concentrations, we achieved the highest producing transfection conditions for DKO and CHO-K1 cells. The optimized transfection conditions for DKO cells required half the amount of DNA and one-third the volume of PEI compared to CHO-K1.…”

Section: Discussionmentioning

confidence: 97%

“…Optimized transfection conditions are essential to maximize transfection yield. Transfection and expression are influenced by different PEI to DNA ratios as it affects the diameter and stability of the DNA:PEI complex . By testing a variety of N:P ratios and DNA concentrations, we achieved the highest producing transfection conditions for DKO and CHO‐K1 cells.…”

Section: Discussionmentioning

confidence: 99%

“…With regards to DNA uptake, it has been shown that the mean diameter of DNA:PEI complexes with lower ratios were larger than those with higher ratios. 39 The fact that DKO and CHO-K1 utilize different optimal PEI and DNA volumes could suggest that variations in plasma membrane composition contribute to the different DNA uptake levels and/or impact the preferences in size and/or charge of DNA:-PEI complexes. However, contrary to our hypothesis, we observed that both cell lines had similar levels of cell surface HS levels and utilized the same initial endocytosis mechanism.…”

Section: Discussionmentioning

confidence: 99%

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Use of an anti‐apoptotic CHO cell line for transient gene expression

Macaraeg¹,

Reilly²,

Wong³

2013

Biotechnology Progress

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Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti-apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro-apoptotic factors, Bax and Bak, in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO-K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ∼3- to 4-fold higher antibody titers than CHO-K1 cells. As evidence of their anti-apoptotic properties post-transfection, DKO cells maintained higher viability and had reduced levels of active caspase-3 compared to CHO-K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post-transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae- and clathrin-mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection-induced apoptosis and not differences in endocytosis mechanism.

show abstract

PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

Cited by 39 publications

References 28 publications

Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Use of a protein engineering strategy to overcome limitations in the production of “Difficult to Express” recombinant proteins

Use of an anti‐apoptotic CHO cell line for transient gene expression

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