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

M. Tomomura, D. S. Rice, J. I. Morgan, Michisuke Yuzaki

Research output: Contribution to journalArticle

65 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)57-63
Number of pages7
JournalEuropean Journal of Neuroscience
Volume14
Issue number1
DOIs
Publication statusPublished - 2001
Externally publishedYes

Fingerprint

Purkinje Cells
Green Fluorescent Proteins
Transgenic Mice
Flow Cytometry
Cerebellum
Neurons
Retinal Bipolar Cells
Fluorescence
Retinal Rod Photoreceptor Cells
Superior Colliculi
Carisoprodol
Dendrites
Automatic Data Processing
Morphogenesis
Nervous System
Axons
Observation

Keywords

  • Cell sorting
  • Cerebellum
  • Green fluorescent protein
  • L7
  • Purkinje cells
  • Retinal bipolar cells

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein. / Tomomura, M.; Rice, D. S.; Morgan, J. I.; Yuzaki, Michisuke.

In: European Journal of Neuroscience, Vol. 14, No. 1, 2001, p. 57-63.

Research output: Contribution to journalArticle

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