Functional Genomics of ATP-Binding Cassette Transporter A3 (ABCA3) Variants.

Functional Genomics of ATP-Binding Cassette Transporter A3 (ABCA3) Variants. Am J Respir Cell Mol Biol. 2020 Jul 21;: Authors: Wambach JA, Yang P, Wegner DJ, Heins HB, Luke C, Li F, White FV, Cole FS Abstract Rare or private, biallelic, variants in the ATP-binding cassette transporter A3 (ABCA3) gene are the most common monogenic causes of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and non-specific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3-/-) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS/GFP/LoxP cassette, fluorescence activated cell sorting (FACS), and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural v...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Am J Respir Cell Mol Biol Source Type: research