Transfection Techniques for Neuronal Cells: Table 1.

Karra, Daniela; Dahm, Ralf

doi:10.1523/jneurosci.0183-10.2010

Cited by 183 publications

(174 citation statements)

References 30 publications

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“…An improved calcium-transfection protocol under ambient CO 2 partial pressure was used to gently transfect mature neurons (Goetze et al, 2004). This technique preserved fine neuronal morphology and survival post-transfection in mature neurons up to DIV21, as observed by us and others (Karra and Dahm, 2010). Transfection efficiencies ranged typically between 5 and 20%, while triplevector co-transfection efficiencies were determined using visibly-expressed fluorescent protein at 6863.84% (n55 wells).…”

Section: Transfection Of Primary Neuronsmentioning

confidence: 91%

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons

König

Fenner

Byrne

et al. 2012

Journal of Cell Science

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SummaryNeuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-kB (NF-kB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/ FGF12), in the regulation of NF-kB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-kB modulator IKKc/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-kB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-kB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-kB activity in neurons.

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Section: Transfection Of Primary Neuronsmentioning

confidence: 91%

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons

König

Fenner

Byrne

et al. 2012

Journal of Cell Science

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“…Whereas efficient plasmid delivery is limited to mitotic cells amenable to chemical transfection or electroporation (Mortimer et al 1999;Karra and Dahm 2010), the ideal CRE-seq delivery vehicle would permit access to a variety of tissues, including post-mitotic tissues, and in a range of species. We reasoned that adeno-associated virus (AAV), a nonpathogenic virus commonly used for gene therapy studies, would be suitable for this purpose.…”

Section: Aav Packaging and Delivery Preserves Cre-seq Library Composimentioning

confidence: 99%

Massively parallel cis-regulatory analysis in the mammalian central nervous system

et al. 2015

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Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.

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“…Several transfection methods for neuronal cells exist, and they are usually grouped into four categories (Karra and Dahm 2010): electrical, chemical, physical and virus-based techniques. Electrical techniques include electroporation and nucleofection.…”

Section: Introductionmentioning

confidence: 99%

Nucleofection of whole murine retinas

2012

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The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.

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Transfection Techniques for Neuronal Cells: Table 1.

Cited by 183 publications

References 30 publications

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons

Massively parallel cis-regulatory analysis in the mammalian central nervous system

Nucleofection of whole murine retinas

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