title: An efficient method for the long-term and specific expression of exogenous cDNAs in cultured Purkinje neurons.
abstract: We present a simple and efficient method for expressing cDNAs in Purkinje neurons (PNs) present in heterogeneous mouse cerebellar cultures. The method combines the transfection of freshly dissociated cerebellar cells via nucleofection with the use of novel expression plasmids containing a fragment of the L7 (Pcp2) gene that, within the cerebellum, drives PN-specific expression. The efficiency of PN transfection (determined 13 days post nucleofection) is approximately 70%. Double and triple transfections are routinely achieved at slightly lower efficiencies. Expression in PNs is obvious after one week in culture and still strong after three weeks, by which time these neurons are well-developed. Moreover, high-level expression is restricted almost exclusively to the PNs present in these mixed cultures, which greatly facilitates the characterization of PN-specific functions. As proof of principle, we used this method to visualize (1) the morphology of living PNs expressing mGFP, (2) the localization and dynamics of the dendritic spine proteins PSD-93 and Homer-3a tagged with mGFP and (3) the interaction of live PNs expressing mGFP with other cerebellar neurons expressing mCherry from a β-Actin promoter plasmid. Finally, we created a series of L7-plasmids containing different fluorescent protein cDNAs that are suited for the expression of cDNAs of interest as N- and C-terminally tagged fluorescent fusion proteins. In summary, this procedure allows for the highly efficient, long-term, and specific expression of multiple cDNAs in differentiated PNs, and provides a favorable alternative to two procedures (viral transduction, ballistic gene delivery) used previously to express genes in cultured PNs.
authors.affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wagner
authors.forename: Seumas
authors.initials: S
authors.lastname: McCroskery
authors.forename: John A
authors.initials: JA
authors.lastname: Hammer
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D018076:DNA, Complementary / Q000378:metabolism*; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D011689:Purkinje Cells / Q000378:metabolism* / Q000821:virology; D013997:Time Factors; D014162:Transfection / Q000379:methods
chemical_list: D000199:Actins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D049452:Green Fluorescent Proteins