title: Establishment of native insulin-negative NOD mice and the methodology to distinguish specific insulin knockout genotypes and a B:16 alanine preproinsulin transgene.
abstract: 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.
authors.affiliation: Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, 4200 East 9th Avenue, B-140, Denver, CO 80262, USA.
authors.forename: Maki
authors.initials: M
authors.lastname: Nakayama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Moriyama
authors.forename: Norio
authors.initials: N
authors.lastname: Abiru
authors.forename: Sunanda R
authors.initials: SR
authors.lastname: Babu
authors.forename: Kamila
authors.initials: K
authors.lastname: Sikora
authors.forename: Marcella
authors.initials: M
authors.lastname: Li
authors.forename: Dongmei
authors.initials: D
authors.lastname: Miao
authors.forename: John C
authors.initials: JC
authors.lastname: Hutton
authors.forename: John F
authors.initials: JF
authors.lastname: Elliott
authors.forename: George S
authors.initials: GS
authors.lastname: Eisenbarth
mesh_terms: D000409:Alanine / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D003433:Crosses, Genetic; D005260:Female; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D007328:Insulin / Q000235:genetics*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008845:Microinjections; D010063:Ovum / Q000502:physiology; D011384:Proinsulin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D019076:Transgenes
chemical_list: D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D011384:Proinsulin; D000409:Alanine