Abstract
Rodent complex trait genetic studies involving a cross between two inbred strains are usually followed by congenic mapping to refine the loci responsible for the phenotype. However, progressing from a chromosomal region to the actual causal gene remains challenging because multiple polymorphic genes are often closely linked. The goal of this study was to develop a strategy that allows candidate gene testing by allele-specific expression without prior knowledge of the credible causal variant. Tnfrsf9 (encoding CD137) is a candidate gene for the Idd9.3 type 1 diabetes (T1D) susceptibility locus in the nonobese diabetic (NOD) mouse model. A C57BL/10Sn (B10)-derived diabetes resistance Idd9.3 congenic region has been shown to enhance accumulation of CD137+ regulatory T cells and serum soluble CD137 in NOD mice. By combining the power of congenic mapping and nuclease-based gene targeting, we established a system where a pair of F1 hybrids expressed either the B10 or NOD Tnfrsf9 allele mimicking coisogenic strains. Using this approach, we demonstrated that the allelic difference in B10 and NOD Tnfrsf9 alone was sufficient to cause differential accumulation of CD137+ regulatory T cells and serum soluble CD137 levels. This strategy can be broadly applied to other rodent genetic mapping studies.
Highlights
Rodent models have been widely used to study the genetics of human complex diseases[1,2]
We previously targeted the second coding exon of the Tnfrsf[9] gene directly in Nonobese diabetic (NOD) mice using a pair of zinc-finger nucleases (ZFNs) to generate the NOD.Tnfrsf9−/− strain lacking CD137 expression[11]
The functional difference has been associated with an increased frequency of CD137+ Tregs in the NOD.Idd9.3B10 strain compared to standard NOD mice[8]
Summary
Rodent models have been widely used to study the genetics of human complex diseases[1,2]. Initial localization of the chromosomal regions is followed by generation of congenic strains to physically map the underlying genes. While these approaches are effective to define a relatively small region harboring phenotype-modulating genetic variants, identification of the causal genes remains difficult. The causal variant could be conclusively determined if coisogenic strains respectively express only one of the two parental alleles of a candidate gene. Compared to NOD mice, the NOD.Idd9.3B10 congenic strain has significantly higher levels of CD137+ FOXP3+ regulatory CD4 T cells (Tregs) and serum soluble CD1378. Recombinant soluble CD137 prevents NOD mice from developing T1D9 These observations provide a possible mechanism of B10 Idd9.3-mediated T1D resistance and support Tnfrsf[9] as its underlying gene. We provide a general strategy for testing candidate genes in rodent genetic mapping studies
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