ArticleMethods in cell biology2016
Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown.
Article in Methods in cell biology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed, 11 citations in OpenAlex.
- Mapping spatial organization of in vitro neuronal networks using high-content imaging.Scientific reports · 2025Article
- Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.JCI insight · 2024Article
- Article
- Myosin 18Aα targets the guanine nucleotide exchange factor β-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.FASEB journal : official publication of the Federation of American Societies for Experimental Biology · 2021Article
- An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons.Cerebellum (London, England) · 2019Article
- Myosin XVI Regulates Actin Cytoskeleton Dynamics in Dendritic Spines of Purkinje Cells and Affects Presynaptic Organization.Frontiers in cellular neuroscience · 2019Article
- Creation of a myosin Va-TAP-tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum.Cytoskeleton (Hoboken, N.J.) · 2018Article
- An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum.Scientific reports · 2017Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture.
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Registered trials
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