discovered using an F2 cohort and a panel of recombinant inbred strains derived from the same two parental strains [33]. Thus, we cannot convincingly conclude at this time that the sex chromo- somes are crucial in mediating trans regulation of gene expression, although our data support this concept. Our finding that 31% of the expressed genes (.6700 transcripts) had a male or female expression bias (confirmed by permutation analysis) lends support for the role of the sex chromosomes in global gene regulation. The large degree of sex bias in gene expression that we detected in macrophages can partly be attributed to the large power we had to detect highly significant sex effects on gene expression even when the absolute effect was small (,20% difference) due to the large sample size (93 female and 114 male samples). This magnitude of sex biased gene expression was previously observed in mouse liver, adipose, muscle, and brain in a similar microarray study using a large F2 cohort [13]. In a study of three pools each of male and female mouse blastocyts using 6 two-dye microarrays, only 600 sex biased genes were detected [18], and we suspect this lower level of detection of sex biased gene expression is primarily due to the small sample size and lower power to detect small effects as significant. It may be argued that some of the sex bias in gene expression in the prior study of mouse liver, adipose, muscle, and brain could be environmental rather than genetic, due to the different hormonal and metabolic environment in male and female mice [12,13]. In contrast, the current study used cells that were grown and differentiated for 2 weeks ex vivo prior to RNA preparation, which should increase the genetic component of the sex bias on gene expression by eliminating the differential and fluctuating hormonal environment of the donor mouse. However, we cannot exclude the possibility of long lasting effects of hormones that may alter cell development and thus gene expression profiles. MATERIALS AND METHODS Mice ApoE-deficient mice [34] on the C57BL/6 genetic background were bred $10 generations onto the AKR/J and DBA/2J genetic backgrounds. A strain intercross was performed using males and females from both parental strains as previously described [35]. The F2 generation mice were sacrificed at 16 weeks of age and bone marrow cells were isolated by lavage of the excised femurs, washed in phosphate buffered saline, plated in Dulbecco’s modified Eagle’s medium (DMEM) with 0.2% BSA into two 100 mm tissue culture dishes and allowed to adhere for 2 hrs at 37uC. Adherent cells were cultured in DMEM with 10% fetal bovine serum and 20% L-cell conditioned media (as a source of MCSF) for 2 weeks at which point they were confluent bone marrow derived macrophages that expressed macrophage specific transcripts [35]. Gene Expression Profiling Total RNA was prepared from macrophages of each mouse using RNAeasy minikits (Qiagen), converted into labeled cRNA, and hybridized to Affymetrix mouse 430v2 oligonucleotide arrays as previously described [35]. MicroArray Suite 5.0 (MAS5.0) software (Affymetrix) was used to compare the 11 perfect matched probes for each element with the mismatched probes and a call of present or absent was made using p,0.05 criteria. The luminosity of each element was normalized to the luminosity of the entire chip. Male and female F2 mice were analyzed separately, since sex is know to play a large role in gene expression levels in mouse tissues [12,13]. Since we were using tissue from a strain intercross, and some transcripts might be absent or low in one of the parental strains, we limited our analysis to transcripts that were called Sex Specific Gene Regulation present for at least 1/3 of the mice for each sex. The sex effect on the level of expression of each probe was compared in the combined female and male cohorts by use of the non-parametric Mann-Whitney U test, and unadjusted and Bonferroni corrected p-values were determined. Genome Scan and eQTL Analysis DNA was prepared from frozen spleen of each F2 mouse and used for SNP genotyping on a 5K mouse SNP chip, as previously described [15], yielding 1967 SNPs on the 19 autosomes and the x chromosome. A polymorphic marker on the Y chromosome, Zfy2, was also genotyped to confirm the grandparental strain of each F2 mouse. Gene expression (not log transformed) and genotype data for each mouse were assembled and analyzed using the r/qtl software package [36], as previously described [15]. To calculate genome wide p-values of the LOD 3.0 and 4.3 thresholds, permutation analysis was performed within r/qtl. Y chromosome LOD scores were derived from the residual sum of squares for the model (rss0) and the residual sum of squares for the Y chromosome effect (rss1), and calculated from the equation LOD = (n/2)6log10 (rss0/rss1), where n = the sample size. Expressed Affymetrix probes on the 430v2 chip were batch queried to NetAffx raw data (release 21) using a custom software application (J. Bhasin, manuscript in preparation) to determine the chromosome and Mb position as well as the % identity for each BLAT alignment of the target sequence for each probe. Probes were scored uniquely mapped to the mouse genome if the % identity of the best match was $75% and the best match had .5% better identity than the second best match. The majority of the uniquely mapped probes had .95% identity with the best match. Probes that failed this test were assigned as ambiguous. Cis and trans eQTLs assignments were restricted to probes that were mapped uniquely. Cis eQTLs were assigned by the same custom software application if a probe’s eQTL was within 20 Mb on the same chromosome as the map position of that probe. All other eQTLs for uniquely mapped probes that did not meet this criteria were assigned as trans eQTLs. eQTLs for ambiguously mapped probes were called ambiguous eQTLs. We observed very high LOD values for several trans eQTLs and did further manual curation that determined these were in fact ambiguous eQTLs due to probe mapping ambiguity. Gene ontology classifications and statistics were performed using GoStat (http://gostat.wehi.edu. au/L) [37]. Data Access Expression and genotype data for each mouse is available in a MIAME compliant format in the Gene Expression Omnibus (NCBI) website, accession # GSE8512. SUPPORTING INFORMATION Table S1 Found at: doi:10.1371/journal.pone.0001435.s001 (2.21 MB XLS) Table S2 Found at: doi:10.1371/journal.pone.0001435.s002 (2.89 MB XLS) Table S3 Found at: doi:10.1371/journal.pone.0001435.s003 (0.02 MB XLS) Table S4 Found at: doi:10.1371/journal.pone.0001435.s004 (0.34 MB XLS) Table S5 Found at: doi:10.1371/journal.pone.0001435.s005 (0.03 MB XLS) PLoS ONE | www.plosone.org 7 January 2008 | Issue 1 | e1435