Aifm1 (R200 del) mice have reduced AIF protein levels across organs and display homogenous phenotypic traits. (A) Schematic representation of the human AIF protein. MLS indicates the mitochondrial localization sequence; FAD and NADH are the FAD-binding and NADH-binding motifs, respectively. Numbers define distinct domains within the AIF protein. Arrows indicate mutated residues identified in patients (in red, R201 deletion). (B) Schematic representation of the genetic targeting strategy, enabling the generation of conditional Aifm1 (R200 del) knockin (KI) as well as Aifm1 knockout (KO) mice. (C) Overexpression of PhiC31-GFP in Aifm1 (R200 del) MEFs resulted in AIF knockout. Green-positive cells displayed loss of AIF staining (red). Hoechst-33342 (blue) was used to visualize nuclei. Scale bar = 20 μm. (D) Aifm1 (R200 del) knockin (KI) mice were born at almost the expected Mendelian ratio. (E) Immunoblot and (F) RT-PCR analyses for Aifm1 expression levels in wt, Aifm1 (R200 del) KI and Hq mutant mice across different organs (for RT-PCR: mean ± SEM, Student's t-test, n = 3–5 per genotype, ***p < 0.001, **p < 0.01, *p < 0.05 compared to littermates). (G–H) Aifm1 (R200 del) KI mice developed (G) hind limb clasping and (H) kyphosis around 6 months of age. (I–J) Hq mutant mice showed high variability in phenotypic traits, such as (I) fur loss and (J) body weight changes (n = 15–20 per genotype); (J) compared to littermate controls, Aifm1 (R200 del) KI mice had a consistent decrease in body weight (n = 18–20 per genotype).