CTLA-4 is a critical "checkpoint" regulator in autoimmunity. Variation in CTLA-4 isoform expression has been linked to type 1 diabetes development in human and NOD mouse studies. In the NOD mouse, a causative link between increased expression of the minor isoform ligand-independent CTLA-4 and a reduction in diabetes has become widely accepted. Altered splicing of CTLA-4 has been attributed to a single nucleotide polymorphism (SNP) in Ctla4 exon2 (e2_77A/G). To investigate this link, we have used NOD embryonic stem (ES) cells to generate a novel NOD transgenic line with the 77A/G SNP. This strain phenocopies the increase in splicing toward the liCTLA4 isoform seen in B10 Idd5.1 mice. Crucially, the SNP does not alter the spontaneous incidence of diabetes, the incidence of cyclophosphamide-induced diabetes, or the activation of diabetogenic T-cell receptor transgenic CD4(+) T cells after adoptive transfer. Our results show that one or more of the many other linked genetic variants between the B10 and NOD genome are required for the diabetes protection conferred by Idd5.1. With the NOD mouse model closely mimicking the human disease, our data demonstrate that knock-in transgenic mice on the NOD background can test causative mutations relevant in human diabetes.