Design, generation, and verification of CNS1-/- in the NOD mouse background.
(A) Schematic representation of the Foxp3 locus highlighting the originally defined CNS1 region (+2003 - +2707 from transcription start site) and corresponding sgRNA sequences for CRISPR/Cas9 targeted cutting. (B) Genotyping PCR showing deletion of the CNS1 region in the primary founder and genetic sequencing after CRISPR/Cas9 deletion verifying a 736 base pair deletion in the Foxp3 locus. (C) CD4+CD8-CD25-CD62Lhi naïve T cells were sorted from NOD and NOD CNS1-/- mice and stimulated in 96-well plates with plate-coated anti-CD3 (2 μg/mL) and anti-CD28 (0.5 μg/mL) antibodies for 72 hours with hIL-2 (100 IU/mL) in the absence or presence of TGF-β. Cells were than stained for CD4, CD25, and Foxp3 and analyzed with flow cytometry (n = 2 mice per genotype, two technical replicates per mouse used).