We hypothesize that NOD mice without native insulin, but with an altered insulin B:9-23 sequence, will be completely protected from diabetes/insulitis if insulin B:9-23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1- and 2-negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR-based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin-negative B:16 alanine transgenic mice have been generated.