Fig. 2

Drosophila Atad3a models show various strength of ATAD3A missense variants. a A schematic of the generation of dAtad3a-T2A-Gal4 by CRISPR-Cas9 gene editing and the translation of a Gal4 protein by a ribosomal skipping mechanism. The location of the attP-SA-T2A-Gal4-polyA-attP cassette insertion into the dAtad3a genomic locus is indicated by the dotted lines. The T2A-Gal4 cassette consists of a splice acceptor (SA, light gray) followed by a ribosomal skipping T2A peptide sequence (pink), a Gal4 coding sequence (green), and a polyadenylation signal (light blue). Two inverted attP sites (blue) are positioned at the 5′- and 3′-end of the cassette. b Expression of UAS-mCD8::GFP under the control of dAtad3a-T2A-Gal4 is monitored in larvae and adult flies. c Complementation test results of dAtad3a-T2A-Gal4 alleles. +, complement; −, failure to complement. dAtad3a-T2A-Gal4 fails to complement a deficiency (Df (3R)Excel7329) that lacks the dAtad3a locus and PBac {PB}dAtad3a^c05496 null allele, which were rescued by the expression of wild-type dAtad3a cDNA. These data indicate that dAtad3a-T2A-Gal4 is a loss-of-function mutant. d Western blots for fly heads expressing wild-type dAtad3a-V5 or dAtad3a-V5 carrying the homologous SNV mutations identified from patients. Five replicates were quantified. Error bars indicate SEM. P values were calculated using Student’s t test. ***P < 0.001. N.S. indicates not statistically significant. e The lethality caused by dAtad3a loss was rescued by the expression of wild-type dAtad3a, and dAtad3a carrying L83V or R176W but not by those carrying F56L, G242V, K574del, or R333P