title: Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown.
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.
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National Institutes of Health, MD, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Alexander
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National Institutes of Health, MD, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Hammer
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000235:genetics; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D006319:Hearing Loss, Sensorineural / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D018995:Myosin Heavy Chains / Q000235:genetics*; D024701:Myosin Type V / Q000235:genetics*; D016116:Piebaldism / Q000235:genetics; D010859:Pigmentation Disorders / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011689:Purkinje Cells / Q000166:cytology*; D014162:Transfection / Q000379:methods*
chemical_list: D035683:MicroRNAs; C513082:Myo5a protein, mouse; D024701:Myosin Type V; D018995:Myosin Heavy Chains
keywords: Cerebellar cultures; L7/Pcp2; Nucleofection; Purkinje neurons; miRNA knockdown