Flow cytometric quantification, Isolation, and subsequent epigenetic analysis of tetraploid neurons

Abstract

Different forms of genomic mosaicism can be detected in vertebrate neurons, including copy number variation, L1 transposition, and aneuploidy. In addition, some populations of vertebrate neurons can also show double the normal amount of DNA, a condition referred to as somatic tetraploidy. These neurons are generated during early stages of development, as they migrate to their adult locations in the adult nervous system, and constitute subpopulations of projection neurons. We have previously shown that neuronal tetraploidy can be characterized by flow cytometry using isolated cell nuclei from different mammalian and avian structures. In this chapter, we describe this procedure using a different model system: the rhombencephalic derivatives from adult zebrafish. In addition, tetraploid neuronal nuclei can be isolated by fluorescence-activated cell sorting and their genomic DNA used for further analyses, either used directly or after whole-genome amplification. Here we show as an example how to perform epigenetic analyses to characterize CpG methylation in differentially methylated regions controlling the Rasgrf1-imprinting domain in mice.