title: Selective and regulated gene expression in murine Purkinje cells by in utero electroporation.
abstract: Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells.
authors: Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors: Jun
authors: J
authors: Nishiyama
authors: Yukari
authors: Y
authors: Hayashi
authors: Toshihiro
authors: T
authors: Nomura
authors: Eriko
authors: E
authors: Miura
authors: Wataru
authors: W
authors: Kakegawa
authors: Michisuke
authors: M
authors: Yuzaki
mesh_terms: D000818:Animals; D018274:Electroporation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D008564:Membrane Potentials / Q000235:genetics; D051379:Mice; D008818:Mice, Neurologic Mutants; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000378:metabolism; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1 / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics; D011689:Purkinje Cells / Q000378:metabolism* / Q000502:physiology; D009435:Synaptic Transmission / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: C498441:Bsn protein, mouse; D009419:Nerve Tissue Proteins; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1