Validating and Optimizing A Combination of INTACT and FACS Techniques for the Isolation of Mouse Astrocyte Nuclei Upon Ablation of the Atrx Intellectual Disability Gene

Abstract

The ATRX gene codes for the ATRX chromatin remodelling protein, which interacts with DAXX protein to deposit histone variant H3.3 at telomeres and pericentromeric heterochromatin. Mutations of the ATRX gene cause intellectual disability. One noticeable example is the ATRX syndrome, which is characterized by distinctive craniofacial features, severe developmental delays, intellectual disability, and mild‐to‐moderate anemia. It is therefore apparent that the ATRX gene likely plays an important role in the central nervous system (CNS). An astrocyte is a cell type that, by the most conservative survey, has the same abundance as neurons in the human brain. However, unlike neurons, astrocyte function is far less understood, and its role other than a supporting function was not known until a decade ago, when astrocytes were discovered to actively participate in higher neuronal processing through the tripartite synapse. The specific role of ATRX in astrocytes has never been reported before. A challenge of elucidating ATRX function in astrocyte is a lack of an optimized technique to isolate a reasonable number of highly enriched Atrx‐ablated astrocyte nuclei for various sequencing purposes, including ChiP‐seq and RNA‐seq. This project aims to fulfill such research demand by validating and optimizing the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique with mouse brain tissue at postnatal day 30. By adjusting various experimental conditions such as tissue homogenization time, the concentration of octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL) in buffer solutions, the dilution ratio of the tissue homogenate and the centrifugation time, a total nuclei yield of 90% was obtained. The structural integrity of nuclei was verified by laser microscopy with nuclei sample stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). However, subsequent enrichment of GFP‐tagged AtrxF/y; Cre+/−;Sun1‐GFP+/− nuclei using anti‐G‐protein coated magnetic bead, as per the original INTACT protocol, is plagued with non‐specific binding of less than 32% specificity. To improve the enrichment factor, a preliminary investigation was performed to validate the use of Florescent‐Assisted Cell Sorting (FACS) to enrich Sun1GFP‐tagged nuclei from the previously‐produced total nuclei preparation. A 92% enrichment of GFP‐tagged nuclei was obtained, demonstrating the promising potential of a combined INTACT‐FACS technique to effectively isolate structurally‐sound nuclei from transcriptionally‐sensitive cell types, within a complex and intermingled tissue environment.Support or Funding InformationThis work was supported by a Dean's Undergraduate Research Opportunity Program award to Yuxuan Jiang and the Canadian Institutes for Health Research MOP#142369.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.