A 20-nucleotide (nt) guide sequence targeting genomic mTOR upstream of Ser1261 was subcloned into pX330. Forward sequence: 5′-caccgacctgccttttggaggttga; reverse sequence: 5′-aaactcaacctccaaaaggcaggac. The following single-stranded oligonucleotide served as repair template, which includes the targeted sequence (underlined), left and right homology arms, and the S1261A mutation (also underlined; AGC to GCG): 5′-gaccctttgatttaccagcatcgaatgctaaggagcagccagggagatgccctggccagtggaccagttgagacaggacccatgaagaaactgcatgtcGCGaccatAaaTctTcaaaaggcaggtccattgtttccagggggattgggaagcagggctctgttttttttctctcattca-3′.

The repair template also included several silent mutations (capitalized) to prevent retargeting of edited genomic signals and a new Nru I restriction site ([tcGCGa]) to facilitate genotyping. The guide RNA (gRNA) targeting plasmid (pX330-mTOR) and the repair template were co-microinjected into single-cell fertilized mouse oocytes and implanted into a pseudopregnant mouse. Heterozygous founders were identified through Nru I restriction of genomic DNA. To confirm co-recombination of both the Nru I site and the S1261A mutation, the genomic region was TOPO-cloned and sequenced.

The following PCR primers were used for TOPO cloning: 5′-GTTGAAATCCTGGCTCTTGC-3′ (forward) and 5′-GCAGGATTTACACGTTTAA-3′ (reverse).

To generate mTORA/A homozygous mice, mice heterozygous for mTOR S1261A were mated (mTOR+/mTORA x mTOR+/mTORA). These mice were generated with the assistance of the Molecular Genetics Core of the MDRC (Michigan Diabetes Research Center) and the University of Michigan Transgenic Core. For genotyping, a ~600-nt fragment of genomic DNA surrounding the mTOR Ser1261 locus was PCR-amplified and digested with Nru I. The following PCR primers were used for genotyping: 5′-GTTGAAATCCTGGCTCTTGC-3′ (forward) and 5′-GCAGGATTTACACGTTTAA-3′ (reverse).

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