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Fig. 1 | Molecular Neurodegeneration

Fig. 1

From: AIF3 splicing switch triggers neurodegeneration

Fig. 1

Identification of a novel AIF isoform-AIF3. a Strategy for conditional deletion of exon3 of mouse Aifm1 gene. A partial restriction map of Aifm1 gene locus and the targeting vector are shown on the first and second lines. Exons of the murine Aifm1 gene are indicated as a filled box and labeled. Exon 3 is flanked by loxP sequences which are indicated by triangles. The G418 resistance gene (Neo) flanked by frt sites is indicated with the open box. The PGK-herpes simplex thymidine kinase (TK) indicated by thick line is inserted at the 5′ end of the targeting vector. Restriction endonuclease sites used for cloning are shown (SphI, EcoRl; XbaI; BglII). Homologous recombination introduces a floxed exon 3 along with the Neo cassette, which is excised by transient overexpression of flpE recombinase in a correctly recombined ES clone. The floxed exon 3 in the Aifm1 allele is further excised by mating with mice expressing Cre recombinase in a brain specific manner. b Expression of AIF3 in different brain regions in AIFfl/Y mice after CamKIIα-iCre recombination at P70 determined by JHU-532 AIF antibody. The recombinant 62 kDa mature AIF (Δ53 aa) and 57 kDa truncated AIF (Δ103 aa), which have an extra 8 aa linker, were used as markers. c mRNA expression and sequence analysis of AIF3 using AIF gene specific primers on its exon 1 and exon 16 (PCR ID: mAIF, Table 2) and cDNA prepared from WT (AIFfl/Y/CamKIIα-iCre-) and AIF3 (AIFfl/Y/CamKIIα-iCre+) mice at P20 as the template. d Establishment of AIF3 MEFs single cell lines from AIFfl/Y/AIFfl/fl mice determined by E1-AIF antibody. e, AIF peptides bridging Exons 1/2, Exons 3/4, and Exons 1/4. f & g MS/MS spectra for AIF and AIF3 peptides bridging Exons 3/4 and Exons 1/4, respectively

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