compound heterozygosity for mutant variants), our findings with the mouse Jak3W81R allele suggest that heterozygosity for dominant negative human JAK3 mutations may cause partially impaired immune response. Although immunodeficiency associ- ated with heterozygosity for a JAK3 mutation has rarely been reported (most of the JAK3 mutations identified to date are homozygous or compound heterozygous)[45,57], our findings raise the possibility that a spectrum of mild immunodeficiency associated with heterozygote JAK3 mutations may be broader than previously suspected. Materials and Methods Ethics statement All mice were maintained under pathogen-free conditions and handled according to the guidelines of the Canadian Council of Animal Care. The experimental protocol (Protocol number 5287) was approved by the ethics committee of McGill University. Mice and parasites Wild type (WT) C57BL/6J (B6), C57BL/10J (B10) and 129S1/ SvImJ (129S1) mice were purchased from the Jackson laboratories (Bar Harbor, Maine, USA). ENU-mutagenized mice were bred at the animal facility of the Goodman Cancer Centre, McGill University. Mice were maintained under pathogen-free conditions and handled according to guidelines of the Canadian Council on Animal Care. P. berghei ANKA parasites were a kind gift from Dr. Mary M. Stevenson, Montreal General Hospital Research Institute, McGill University. Parasites were maintained as frozen stocks at 280uC, and passaged weekly in B10 mice (donor mice). Infected B10 mice were monitored daily for parasitemia and when parasitemia reached 4–7%, mice were bled, and the blood was diluted in 16 phosphate-buffered saline (PBS) for infection of ENU-mutagenized mice. ENU Mutagenesis and breeding Twenty 8-week-old WT B6 male mice were mutagenized by intraperitoneal injection of a fractionated dose of 3690 mg/kg of ENU at 1-week intervals. After recovery of fertility (8–15 weeks post treatment), treated males were used in a breeding scheme designed to uncover recessive mutations as previously described [59]. Briefly, treated males (G0) were bred to WT B10 females to generate G1 animals, which are heterozygous for mutations across their genome. G1 males were crossed to B10 females to generate G2 animals, each of which has a 50% chance of inheriting any single mutation carried by their G1 father. Two G2 females were backcrossed to their G1 father to generate G3 animals, about a quarter of which were expected to be homozygous for mutations carried by the G1 male. In order to introduce a higher degree of polymorphism in the offspring to facilitate genetic mapping, G1 males from pedigrees with a confirmed heritable resistance trait (after phenotyping of G3 animals) were out-crossed to 129S1 female mice to generate F1 animals. F1 mice were intercrossed to generate F2 animals, 25% of which were expected to carry the mutation from the G1 male fixed to homozygosity (Figure 1A). Infection with Plasmodium berghei ANKA G3 and F2 mice at $7 weeks of age were infected intravenously (i.v.) with 106 P. berghei ANKA-parasitized erythrocytes (obtained from parasite donor mice), and were monitored 2–3 times daily for the appearance of characteristic neurological symptoms, for weight loss and for survival. Mice that survived greater than 13 days post infection with no neurological symptoms were considered to be resistant to cerebral malaria. B10 and 129S1 A Jak3 Mutation Protects against Cerebral Malaria mice were used as susceptible controls in all experiments, while IFN-c knockout (KO) mice (on a B6 background) were used as resistant controls. DNA preparation and Genetic Mapping Tail biopsies were obtained from all mice, and genomic DNA was isolated by a standard procedure using proteinase K digestion and phenol/chloroform extraction, as previously described [60], and DNA samples were diluted to 20 ng/ml in distilled H2O for genotyping. Genome scanning was performed at the McGill University and Genome Quebec Innovation Centre (Montreal, Qc, Canada), using DNA samples from 15 resistant and 29 susceptible G3 mice from pedigree 48 (P48), and the massArray platform from Sequenom with a panel of 131 B6/B10 polymor- phic markers (SNPs) distributed across the genome. Mapping data were analyzed with the R/qtl software version 2.10.1. The binary model was used, and LOD scores calculated using survival as a phenotype. The cutoff for genome-wide significance (p,0.05) was 3.23. Further linkage analysis was conducted in 211 additional (P48 X 129S1) F2 mice (see Figure 1A) genotyped for microsatellite markers (Mouse Genome Informatics Database; www.informatics.jax.org) informative for B6 and 129S1 progen- itors. Genotyping was carried out by a standard PCR-based method using [a-32P] dATP labeling, and resolution on denaturing 6% polyacrylamide gels. Genome Sequencing and analysis The genome of the P48 mutant homozygotes was sequenced using whole-genome shotgun sequencing with 8 lanes of the Illumina GAII sequencer, yielding 133 million 76bp reads (9.1 Gbases in total, corresponding to a 36 coverage of the genome). Sequence extraction and base calling was done using Illumina Pipeline software. Reads were trimmed at five base running median quality value Q = 20 and aligned to the reference genome (mm37) using Bowtie (PMID: 19261174) and BWA (PMID: 19451168). Variant calling was performed using SamTools (PMID: 19505943) and results from both mapping methods were merged after variant calling, producing a total of 350,607 candidate variants. After filtering out non-homozygous and low coverage SNPs (less than 36) and retaining only coding non- synonymous/non-sense mutations using the ENSEMBL’s variant effect predictor, this number was reduced to only 831 variant candidates. Only 6 of those were located in the 17 Mb region of chromosome 8 to which the mutation had been mapped, including 2 candidate mutations in the Jak3 gene. Immunophenotyping Following isolation of cells from different tissues (spleen, thymus, bone marrow, lymph nodes and blood), the cells were surface stained with appropriate dilutions of antibodies (determined from titration experiments), for 20 minutes in the dark at 4uC, fixed in PBS containing 1% formaldehyde and stored at 4uC in the dark until FACS analysis (performed within 24 h). The following anti- mouse monoclonal antibodies were used: FITC anti-CD4 (RM4- 5), PE anti-CD8a (53-6.7), PECy7 anti-CD19 (1D3), APC anti- CD11c (HL3), APCCy7 anti-GR1 (RB6-8C5), V450 anti-CD117 (2B8) (all from BD Pharmingen); PerCPCy5.5 anti-F4/80 (BM8), PerCPCy5.5 anti-CD3e (145-2C11) and eFluor 450 anti-CD11b (MJ7/18) (all from eBioscience). A minimum of 105 cells was collected by FACS for each tissue sample. Data analysis was performed using FACS DiVa version 6.0 software. Initial gating of each sample set used a forward scatter (FSC)-area versus an FSC- height plot to gate out cell aggregates. Immune cells were isolated, and the different cell populations stained with various antibodies PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31012