File S1 - Generation of TALEN-Mediated GRdim Knock-In Rats by Homologous Recombination

Supplementary Data are available at PLoS ONE Online and include Table S1–S2 and Figure S1–S5. Table S1: Nr3c1 sequence for TALEN design and binding site sequences. Table S2: Primers used in this experiment. Figure S1 Evaluation of TALEN expression in C6 rat cells. C6 cells were transfected with either TAL 3, 6 or 13, Right (R), Left (L) or both (RL). Western blot was performed with antibody against flag tag. Mcells are mock-transfected cells. Figure S2 Sequence of the Nr3c1 gene donor plasmid. The homology arms are indicated in purple, the exons and introns of the Nr3c1 gene are indicated in orange and black lines respectively. TALEN binding sites are indicated in blue and the spacer in red. Nucleotides that are mutated in the donor are marked in bold letters. HaeIII site in the donor is underlined in blue. Suppressed AluI site (is underlined in purple). Figure S3 Gel digestion analysis of the 3.4 rat Nr3c1 exon 3. Primer pairs used in this experiment (“outside-in”) are shown in Table S2 in File S1. Wt indicates expected fragments of wild type animals for both AluI and HaeIII enzymes. The stars indicate bands expected of the donor sequence: for AluI digestion: one upper band of 883 bp (*), for HaeIII digestion: two bands of 524 (**) and 359 bp (***). Rat 3.4 also shows wt digestion pattern, indicating that it is heterozygous for the pA476T mutation. Figure S4 Sequencing of the Nr3c1 gene in 3.4 KI rat. Genomic DNA was amplified with primers outside out the donor sequence (“outside-out”) (c. f. Table S2 in File S1), and cloned into TOPO cloning vector. 21 clones were selected for sequencing. Here we show 3 representative sequences. Wt, wild type sequence; exp, expected sequence, DNA1, clone 1, DNA2, clone 2, DNA5, clone 5. TALEN binding sites from the donor are in green, bold letters indicate the mutated nucleotides. HaeIII site is present in the donor sequence only; AluI is present in the wild type sequence only. Figure S5 Gel digestion analysis of “indels” in Fo founders rats. “Inside-out” primers were used in this assay (Table S2 in File S1). Wt indicates expected fragments of wild type animals upon digestion with AluI enzyme. All pups have variable size bands on the lower molecular weight zone, indicating possible indels. The KI rat 3.4 upper band (marked with a * of 469 bp) indicates absence of the AluI site close to the pA476T. KI fragment sizes expected: 125, 250 and 469 bp Wt fragment sizes expected: 125, 175, 250 and 294 bp. (DOCX)