A flow cytometry-based method for gene expression profiling of CNS cell types

Abstract

In this thesis, I present a technique for profiling gene expression of specific cell types in the central nervous system (CNS), called fluorescence activated nuclei sorting (FANS). FANS utilizes flow cytometry to isolate cell nuclei from CNS subtypes and microarray analysis of nuclear mRNA. When compared to an existing technique, translating ribosome affinity purification, it was found that FANS was able to detect differentially expressed genes between two types of medium spiny neurons in the striatum with similar or higher sensitivity, using transgenic mice with fluorescence proteins labeling the desired cell types. Immunofluorescence experiments were also performed to label cell nuclei isolated from wild type mouse CNS for FANS analysis. It was found that our staining method successfully labeled neuronal nuclei using a NeuN antibody, but did not label enriched markers of medium spiny neurons. Further studies are needed to increase the signal-to-back-ground ratio of these stainings, which would allow FANS to be applied to wild type animals.