Although a very small number of Daporinad purchase non-Purkinje cells were sometimes EGFP-positive, they were always negative for DsRed2 (Fig. 3D, a–c). The only DsRed2

signals observed outside the cerebellum were within the dorsal cochlear nucleus (Fig. 3D, d). Indeed, cartwheel cells in the dorsal cochlear nucleus are known to share several cell markers, such as calbindin and L7, with Purkinje cells, and cartwheel and Purkinje cells are probably derived from common precursors (Berrebi et al., 1990). Together, these results indicate that IUE can drive the expression of exogenous genes specifically in Purkinje cells in a temporally controlled manner, by using the L7 promoter and inducible Cre/loxP system. As shown by the successful application of an inducible Cre/loxP system consisting of three plasmids (Fig. 3A), a major advantage of the gene delivery by in vivo electroporation is that

multiple and very large genes can be coexpressed with high efficiency (Saito & Nakatsuji, 2001; Matsuda & Cepko, 2007; Barnabe-Heider et al., 2008). To further confirm this principle in our system, we electroporated at E11.5 three plasmids encoding three different fluorescent proteins: mito-ECFP, which is designed to localize to mitochondria, EGFP-β-actin (Furuyashiki et al., 2002) and DsRed2. The confocal z-stack images of spectral data were obtained on Forskolin mouse fixed sagittal sections at P14, and the individual ECFP, EGFP and DsRed2 fluorescence images were separated by the linear unmixing method (Zimmermann et al., 2003). Most Thiamet G labeled Purkinje cells (99.1%; 445 of 449 cells) expressed all three fluorescent proteins (Fig. 4).

The DsRed2 signals were observed diffusely throughout Purkinje cells, including the soma, dendrites, spines and axons. In contrast, the EGFP-β-actin signals accumulated in the dendritic spines and nuclei, while the mito-ECFP signals were observed in the soma and dendritic shafts. Next, to examine whether a large gene can be introduced into Purkinje cells by IUE, we used cDNA encoding Bassoon, a large protein selectively localized at the active zone of presynaptic nerve terminals (tom Dieck et al., 1998). We electroporated a plasmid (approximately 17 kb) encoding mouse Bassoon fused to mCherry (mCherry-Bassoon; approximately 12.5 kb) and a plasmid encoding EGFP at E11.5. Confocal imaging of fixed cerebellum at P14 revealed punctate mCherry-Bassoon signals along EGFP-positive Purkinje cell axons (Fig. S4). In addition, mCherry-Bassoon signals were colocalized with immunoreactivity for vesicular GABA transporter (VGAT), a presynaptic marker (Fig. S4). Together, these results illustrate that an advantage of IUE-based gene delivery into Purkinje cells is that not only can multiple genes be coexpressed, but also that large genes can be transfected with high efficiency.