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

Lin, Peng; Xiong, Wendian; Cai, Yanfei; Chen, Yun; He, Yao; Yang, Jianfeng; Jin, Jian; Li, Huazhong

doi:10.1080/21655979.2016.1222995

Cited by 14 publications

(16 citation statements)

References 20 publications

(30 reference statements)

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“…Filler DNA is both cost‐attractive and can accelerate the process by enabling the production of broad panels of hit sets from small‐scale high‐throughput plasmid preparations. Several publications [ 25,26,27 ] have demonstrated that the addition of irrelevant DNA as Filler or carrier to the transfection process can improve transfection efficiency, alter transcription rates and ultimately increase the amount of protein obtained. Although several mechanisms of action have been proposed, it is not fully understood which mechanisms lead to these benefits.…”

Section: Discussionmentioning

confidence: 99%

Optimization of a transient antibody expression platform towards high titer and efficiency

Greene

Cazacu

Tamot

et al. 2021

Biotechnology Journal

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Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs.

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Section: Discussionmentioning

confidence: 99%

Optimization of a transient antibody expression platform towards high titer and efficiency

Greene

Cazacu

Tamot

et al. 2021

Biotechnology Journal

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“…In most studies, transfection effi ciency is evaluated using biomarkers such as luciferase and GFP (7,8,10,11,17,18). In this study, GFP in pEGFP-N1 and pCDH plasmid vectors were used as strong biomarkers for evaluating fl owcytometry.…”

Section: Discussionmentioning

confidence: 99%

Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents

Rahimi¹,

Mobarakeh²,

Kamalzare³

et al. 2018

BLL

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OBJECTIVES: In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identifi ed. BACKGROUND: Lipofectamine 3000 is a new transfection reagent which is claimed to be more effi cient than other transfection reagents like Turbofect. Transfection effi ciency could be affected by the nature of target cell line and vector. METHODS: Transfection effi ciency and cytotoxicity of each reagent was identifi ed by using fl ow cytometry and XTT assay, respectively. RESULTS: Lipofectamine 3000 was more effi cient in transfecting pCDH, while Turbofect was more effi cient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not suffi ciently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in suffi cient transfection. CONCLUSION: Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).

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“…Albeit these reporter genes are often used interchangeably in transfection experiments, each transgene and respective assay has a different sensitivity and its own metrics [161]. For instance, fluorescent proteins are good descriptors of the transfection efficiency at the single cells level, which is typically defined as the percentage of cells expressing the transgene-encoded protein [162][163][164]. Conversely, luciferase expression provides relevant information about pDNA expression levels within a cell population but not in single cells, as the chemiluminescence is expressed as arbitrary luminescence units per milligram of proteins in cell lysates [165][166][167][168].…”

Section: Evaluation Of Transfection Effectiveness: a Trade-off Betweementioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

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

Cited by 14 publications

References 20 publications

Optimization of a transient antibody expression platform towards high titer and efficiency

Optimization of a transient antibody expression platform towards high titer and efficiency

Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

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