High-Throughput Transfection of Differentiated Primary Neurons from Rat Forebrain

Marine, Shane; Freeman, Jamie; Riccio, Antonella; Axenborg, Marie-Louise; Pihl, Johan; Ketteler, Robin; Aspengren, Sara

doi:10.1177/1087057112439233

Cited by 8 publications

(5 citation statements)

References 17 publications

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“…It was shown that the capillary electroporation concept of the Cellaxess Elektra allows the successful transfection of hippocampal and cortical neurons, which were isolated from rat brains (E18) and subsequently cultured for 5 d to allow axon and dendrite development. 72 In this study, two short 2 ms pulses at 350 V resulted in 30% of GFP-positive neurons when a GFP plasmid was used for transfection and a significant reduction of GFP when siGFP was co-transfected. Cell viability was not impaired compared with nonelectroporated samples.…”

Section: Applications Of Electroporation In Htsmentioning

confidence: 75%

Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues

Luft

Ketteler

2015

SLAS Discovery

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The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

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Section: Applications Of Electroporation In Htsmentioning

confidence: 75%

Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues

Luft

Ketteler

2015

SLAS Discovery

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“…Methods for delivery of siRNA/shRNA include lipofection--based transfection, electroporation and lentiviral transduction 59,60 . Most cell types can be transfected reasonably well with one of these methods.…”

Section: Interactome Profiling: Ppi--approachesmentioning

confidence: 99%

Application guide for omics approaches to cell signaling

et al. 2015

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Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single--gene approach does not take into account the whole complexity of cell signaling. With the availability of OMICs--techniques, great progress has been made in understanding signaling networks. OMICs approaches can be classified into two categories: "molecular profiling", including genomic--, proteomic--, post--translational modification--and interactome--profiling; and "molecular perturbation", including genetic and functional perturbations.Due to the ever--growing field of method development to characterize cellular processes on genomic and proteomic levels and in many other dimensions, it has become a challenge to select and apply the appropriate methods suitable for addressing a specific biological question. In this perspective, we will describe some selected OMICs--techniques and discuss their applicability to cell signaling research.3

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“…42, 79, 107 Thus, the voltage, pulse shape, pulse duration, nucleic acid quantity, and cell density must be experimentally determined for each cell type to achieve maximum transfection efficiency, cell viability, and gene expression. 74, 104, 162 Mehier-Humbert et al . 108 suggested that long pulses (20–60 ms) combined with modest field strengths (100 – 200 V/cm) produce larger pores in cell membranes that remain open for longer durations.…”

Section: Physical Gene Delivery Strategiesmentioning

confidence: 99%

Physical Non-Viral Gene Delivery Methods for Tissue Engineering

2012

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The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

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High-Throughput Transfection of Differentiated Primary Neurons from Rat Forebrain

Cited by 8 publications

References 17 publications

Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues

Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues

Application guide for omics approaches to cell signaling

Physical Non-Viral Gene Delivery Methods for Tissue Engineering

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