Purification of Purkinje cells by fluorescence‐activated cell sorting from transgenic mice that express green fluorescent protein

Abstract

The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.

Scotopic Visual Signaling in the Mouse Retina Is Modulated by High-Affinity Plasma Membrane Calcium Extrusion

“…Cones do not express PMCA2, suggesting the cone pathway is dominated by the less sensitive PMCA1 mechanism (Križaj et al, 2002;Haverkamp et al, 2003). A subset of GFP-negative INL cells in retinas from L7-GFP transgenic mice was immunoreactive for PMCA2, indicating that PMCA2 could be localized to cone bipolar cells (Tomomura et al, 2001). However, our observation that photopic ERG is unaffected in PMCA2-deficient mice, together with lack of PMCA2 expression in cones, suggests that this isoform does not play a major role in the cone pathway.…”

“…4C,D). Immunostaining of retinas from transgenic mice that express high levels of GFP in rod bipolar cells (Tomomura et al, 2001) additionally suggested postsynaptic localization of PMCA2 to rod bipolar cells (Fig. 4C, arrowheads).…”

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination

“…To confirm this, we used transgenic mice expressing EGFP under a Purkinje cell-specific promoter, Pcp2 (25). We FACS-sorted their EGFP-positive Purkinje neurons from other cell types in a cerebellar suspension (25) and Western blotted both populations for P-Rex2 expression. Indeed, P-Rex2 protein was found specifically in Purkinje cells (Fig.…”

Uses of GFP in Transgenic Vertebrates

“…GFP transgenic animals can also be used to mark cell types of interest to be purified by fluorescent activated cell sorting (FACS). Transgenic mice expressing GFP under the control of the L7 promoter were used to purify live Purkinje cells (Tomomura et al, 2001). A knock-in of GFP into the Hoxa13 locus in mice was used to purify limb mesenchymal cells which were then used to show Hoxa13 null cells are defective in forming chondrogenic condensations in vitro (Stadler et al, 2001).…”