Figure 1. An ENU mouse mutant with increased CD44hi CD4 cells and anti-nuclear autoantibodies.

(A and B) Representative flow cytometry showing on peripheral blood CD4⁺ cells (A) CD44 expression with the gate used to define CD44^hi cells and (B) FOXP3 vs CD44 phenotype including normalized CD44 Mean Fluorescence Intensity (MFI) of the gated CD44^lo FOXP3⁻ subset from Rasgrp1^+/+ (WT), heterozygous Rasgrp1^Anaef/+ or homozygous Rasgrp1^Anaef/Anaef mice. Statistical analysis (right) used unpaired Student’s t tests where each symbol represents an individual mouse; ***p<0.001. (C) Antinuclear antibodies (ANA) in diluted blood plasma from a B6xB10. Rasgrp1^Anaef mouse and wildtype littermate, measured by indirect immunofluorescence on HEp-2 cells. Note homogeneous nuclear staining of interphase cells and positive chromatin bars in dividing cells (marked with arrow). Magnification 20 ×. (D) Quantitation of positive ANA results for wildtype, Rasgrp1^Anaef/+ and Rasgrp1^Anaef/Anaef C57Bl/6xC57Bl/10 siblings tested at 15 weeks of age.

DOI: http://dx.doi.org/10.7554/eLife.01020.003

Figure 1—figure supplement 1. Mapping and genotyping of the Rasgrp1 mutation in ENU mutant mice with anti-nuclear antibodies and CD44hi phenotype.

(A) SNP analysis of a cohort of F2 mice generated by outcrossing a B6 animal identified as ‘affected’ with CBA, then incrossing F1 offspring to produce the (ENU.B6 × CBA/J) F2 animals listed. Phenotypes of affected mice tracked with a genomic region between 114–121.2 on chromosome 2. The Rasgrp1 gene lies in this interval. (B) Genotyping of Rasgrp1^Anaef/Anaef, Rasgrp1^Anaef/WT, and wildtype mice in the Roose lab was performed using MS-PCR as described (Rust et al., 1993; Bottema and Sommer, 1993). Products are wt: 202 bp and R519G: 223 bp. (C) Genotyping of Rasgrp1^Anaef/Anaef, Rasgrp1^Anaef/WT, and wildtype mice using Amplifluor PCR in the Goodnow Lab. The Rasgrp1^Anaef strain was established through ethylnitrosourea (ENU)-mediated mutagenesis of B6 mice at the Australian National University as previously described (Randall et al., 2009).

DOI: http://dx.doi.org/10.7554/eLife.01020.004