Fig. 1.
Generation of editing-defective AlaRS mouse models. (A, Left) Misacylation of tRNAAla with serine by mouse wild-type (WT) or C723A AlaRS. (Right) Relative level of mischarged Ser-tRNAAla in the presence of WT, C723A, or equal molar ratio of WT and C723A AlaRS at 15 min. Assays were run in duplicate, and points (mean ± SD) were averaged from two experiments. (B) To generate the conditional knock-in allele of AlaRSC723A, a loxP-flanked transcriptional stop cassette was inserted in intron 1, and the TGT codon encoding cysteine at position 723 in exon 15 was replaced with a GCG alanine codon. Aars transcription remains off until the stop cassette is deleted via Cre expression. The Southern blot probe (5′ probe), the size of the left and right “arm” of the targeting vector, and the distance between the neo cassette and exon 15 are indicated. (C) Body weight of WT (n = 13), Aarssti/sti (sti/sti; n = 8), and Aarsstop/sti (stop/sti; n = 11) mice at postnatal ages (P) 14 and P21. Values represent mean ± SD; ***P < 0.0001 (one-way ANOVA). (D) Photograph of 2-mo-old WT, Aarssti/sti, and Aarsstop/sti mice. Note the dorsal alopecia characteristic of the Aarsstop/sti mouse.