Quality control and protocols
Phenotypic Quality Control
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Incidence Studies
Because environment is a potent modulator of type 1 diabetes (T1D) gene penetrance, the T1DR will determine if the reported ability of a stock, and the particular genetic modification, to modulate T1D is retained under full barrier health status. Incidence studies will be performed to determine frequency and rate of mice of each sex, as well as NOD/LtJ.
Genetic Quality Control
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Genome Scans
For stocks in which a congenic interval, transgene or gene knockout has been introduced from another strain, we conduct genome-wide scans to verify the exclusion of unwanted donor strain material. We analyze on average 8 markers per chromosome, which are spaced approximately 10-20 cM apart and delineate NOD, 129, and C57BL/6 origin in the stock's genome. -
Verification of markers at known diabetes susceptibility/resistance (Idd) loci
As an extension of the genome scan, we concentrate on all reported diabetes susceptibility/resistance loci in a separate genomic microsatellite analysis for stocks in which a congenic interval, transgene or gene knockout have been introduced from another strain into NOD (or NOD genome introduced into a diabetes resistant background). The markers used allow allele distinction between most donor and recipient strains. It should be noted that not all Idd susceptibility alleles are NOD in origin (e.g., Idd7 on Chromosome 7 and Idd14 on Chromosome 13). -
Chromosome of Interest Analysis
For stocks in which a congenic interval, transgene or gene knockout have been introduced from another strain, a series of microsatellite markers will be genotyped to determine the extent of donor strain genome in the flanking regions of the target gene or segment. As most mutations are currently made using embryonic stem (ES) cells from 129 substrains, there is always 129 genome carried over in linkage disequilibrium with the targeted allele. The length of the congenic segment can be in excess of 20 cM, even after 10 backcrosses with selection only for the targeted allele. In certain instances, 129 genes in the congenic interval rather that the targeted gene can be responsible for a reduced diabetes frequency in the congenic stock.
Immunology protocols
- Dr. Ed Leiter contributed a chapter to Current Protocols in Immunology (1997) (.pdf) 15.9.1-15.9.23, which we have reproduced here by permission of the editor, John Wiley & Sons, Inc.
If you have any questions regarding any of our studies, please contact the Project Manager