The strain-specific difference in <i>Itgal</i> message level is determined by <i>cis</i>-acting elements at the <i>Civq1</i> locus.

<p>A. mRNA levels of the canonical isoform of <i>Itgal (Itgal-002)</i> in adult cortex from B6, BALB/c, SWR, and CSS7 strains as determined by qRT-PCR. Each bar represents mean ± SEM. ** <i>P<0.001</i> vs. B6. B. The allele-specific expression ratio of <i>Itgal</i> transcript (<i>Itgal</i>-002) levels in F1 (B6×BALB) mice. A coding SNP (rs107634043) in exon 30 was used to monitor the B6-allele and BALB/c-allele transcripts of <i>Itgal</i> in macrophages (MΦ), endothelial cells (EC), P1 and the adult cerebral cortices. The level of the B6-specific <i>Itgal</i> transcript was approximately 6-fold higher than BALB/c-transcript in adult cortex. Each bar represents the allele-specific expression ratio (B6/BALB) averaged for three F1 animals. C. Surface expression of CD11a (ITGAL) on CD11a+ brain immune cells. CD45+ CD11a+ brain cells from BALB/c mice express significantly less surface CD11a as assessed by flow cytometric staining than B6 CD45+ CD11a+ brain cells (** <i>P<0.001</i>).</p

Infarct volume is increased in <i>Itgal</i> knockout (KO) mice.

<p>A. Collateral number does not differ between B6 and <i>Itgal</i> KO mice. B. The posterior faces of representative 1-mm coronal sections from B6 and <i>Itgal</i> KO mice are shown. The size of the infarct in <i>Itgal</i> KO is larger than B6 mice. C. <i>Itgal</i> KO mice on the B6 background exhibit infarct volume ∼3-fold larger than that of B6 mice (15.9 vs. 4.7 mm³). ** <i>P<0.001</i>. D. Increased neuronal death in the cortical region of the brain slices from <i>Itgal</i> KO mice compared to control B6 mice 24 hr after 5.5 min OGD. E. Deficiency of <i>Itgal</i> increases neuronal cell death by ∼50% compared to B6 mice. Values represent mean ± SEM from at least 5 animals per group. ** <i>P<0.001</i>.</p

The critical interval of chromosome 7 retains both infarct volume and collateral artery number phenotypes in congenic animals.

<p>A. Schematic map of two <i>Civq1</i> congenic lines carrying segments of B6 chromosome 7 introgressed into the BALB/c background, and heterozygous BALB/c alleles on B6 background. The latter could only be maintained as a heterozygote with B6. B. The posterior faces of representative 1-mm coronal sections from the B6, C.B6-<i>Civq1</i>-3, and BALB/c mice are shown. The size of the infarct is much smaller in C.B6-<i>Civq1</i>-3 than control BALB/c mouse. C. Congenic mice (C.B6-<i>Civq1</i>-1 and -3) exhibit a reduction in infarct volume by ∼30% compared to BALB/c mice. Infarct volume in congenic mice carrying heterozygous BALB/c alleles on B6 background (B6.C-<i>Civq1</i>(HET)) is 2.5-fold larger than that of control B6 mice. ## <i>P<0.001</i> vs. BALB/c; *<i>P<0.05</i>, ** <i>P<0.001</i> vs. B6. Values represent mean ± SEM. D. Superficial angioarchitecture of the brain in 4-week old B6, C.B6-<i>Civq1</i>-3, and BALB/c mice. Magnified images of the box in the upper panels are shown in the lower panels. The red arrowheads indicate the vessels of pial anastomoses between the MCA and ACA. E. C.B6-<i>Civq1</i> congenic lines show an increase in the number of collaterals connecting the distal MCA and ACA compared control BALB/c strains. Heterozygous congenic mice show no difference compared to control B6 mice. ## <i>P<0.001</i> vs. BALB/c; ** <i>P<0.001</i> vs. B6. F. Cortical brain slices from B6, BALB/c, and C.B6-<i>Civq1</i>-3 mice biolistically transfected with an YFP expression plasmid under normal conditions and 24 hr after 5.5 min of OGD. G. C.B6-<i>Civq1</i>-3 mice exhibit ∼50% increased neuronal viability compared to control BABL/c mice. Total numbers of healthy and YFP-positive neurons in the cortical region of the brain slices were counted at 24 hr after transient OGD. Cell viability was normalized from their non-OGD controls. Values represent mean±SEM from at least 5 animals per group. * <i>P<0.05</i> and ** <i>P<0.001</i>.</p

Oxygen deprivation increases cleaved Caspase 3 in an <i>ex vivo</i> brain slice stroke model.

<p>A. Efficiency of <i>Itgal</i> knock-down in <i>ex vivo</i> brain slices using siRNA. Non-target or <i>Itgal</i>-specific siRNA were transfected into cortical brain slices from B6 mice for 3 days. <i>Itgal</i> mRNA levels normalized to <i>Gapdh</i> control were determined by qRT-PCR. Values represent the mean±SEM of 4 mice.***<i>P<0.01</i>. B. Western blots were performed for cleaved Caspase-3 in explanted brain slices transfected with either non-target siRNA or <i>Itgal</i>-specific siRNA in both control and oxygen deprivation (OD) conditions. C. Quantification of panel B. Cleaved Caspase-3 expression level was normalized to an alpha-tubulin control. Values represent the mean±SEM of 4 mice. *** <i>P<0.001</i>.</p

<i>Civq1</i> on chromosome 7 re-identified in intercrosses between multiple mouse strains.

<p>A. Genome-wide EMMA analysis across chromosome 7 for cerebral infarct volume in 32 inbred mouse strains. The plot is drawn for −log(P) against SNP positions on the chromosome. Green crosses represent SNPs over the significance threshold of P = 10⁻⁵. The genomic region (132.35–134.81 Mb) with significant association in chromosome 7 maps within <i>Civq1</i> detected by multiple linkage studies. B. The graph presents the results of a genome-wide linkage scan for infarct volume in 35 (FVB×BALB) F2 progeny. Chromosomes 1 through X are represented numerically on the x-axis and the y-axis represents the LOD score. The significant (<i>P<0.05</i>) level of linkage is determined by 1,000 permutation tests. Only a single genomic region on chromosome 7 displays significant linkage to infarct volume with a LOD score of 5.2. C. <i>Civq1</i> is mapped to distal chromosome 7 by linkage analyses in multiple F2 (B6×BALB/c, B6×SWR, and FVB×BALB/c) populations. The same SNP marker (rs13479513) is located at the peaks of all linkage analyses.</p

Fine mapping of <i>Civq1</i> harboring a causative candidate gene for infarct volume.

<p>The map shows chromosome 7 with the previously mapped QTLs and their 95% confidence intervals, CSS7, the critical congenic interval, the genome-wide association peak region, and the interval-specific SNP haplotype block analysis among the 5 mapping strains. The 4 candidate genes are clearly located with SNP haplotype blocks shared between the small infarct strains (B6 and FVB) but differing from the large infarct strains (BALB/c, SWR/J, and A/J).</p

A truncated cytoplasmic tail of the protein and increased mRNA level of a splice variant of <i>Itgal</i> correspond with allelic variation between mouse strains.

<p>A. Schematic of the <i>Itgal</i>-003 transcript showing sites of the ∼150-bp deletion in intron 29 and a 5-bp insertion (TTCT and A) in the coding region of exon 30b, present in BALB/c, SWR, and A/J strains. The 5-bp insertion causes a premature termination of the protein, resulting in a shorter cytoplasmic tail of the protein. B. Allelic variation of the ∼150 bp deletion in intron 29 in 5 mapping strains is confirmed by PCR of genomic DNA, with primer pairs (white arrow in A). C. Amino acid sequences of the cytoplasmic tail of the <i>Itgal</i>-003 protein. The 5-bp insertion in exon 30b results in an <i>Itgal</i>-003 protein that is identical to the canonical protein, except a short stretch of new amino acid sequence in the truncated cytoplasmic tail. The 5-amino acid sequence in red is matched with the electropherograms in (A). The 19 amino acids (GQRRDIGMDQEERGPGRL) bolded and underlined are used as an epitope to generate an <i>Itgal</i>-003-specific polyclonal antibody. D. mRNA levels of <i>Itgal</i>-003 transcripts (ENSMUST00000120857) in P1 cortex from B6, FVB, BALB/c, SWR/J, and CSS7 strains as determined by qRT-PCR. Each bar represents mean ± SEM. E. The allele-specific expression ratio of <i>Itgal</i>-003 in F1 mice. The level of BALB/c-specific <i>Itgal</i>-003 transcript was approximately 26 times, 44 times, and 19 times higher than the B6-specific transcript in macrophages (MΦ), endothelial cells (EC) and P1 cortex, respectively. <i>Itgla</i>-003 was not detected in adult cortex (ND). Each bar represents the allele-specific expression ratio (BALB/B6) of the <i>Itgal</i> transcript averaged for three F1 animals. F. Western blot for an epitope specific to the <i>Itgal</i>-003 encoded protein. Lysates of P1 brain of B6, BALB/c, <i>Itgal</i> knockout (KO) and FVB mice. Cos (+), the recombinant <i>Itgal</i> protein generated by transfection of COS-7 cells with <i>Itgal</i>-003 cDNA and Cos (−), MOCK transfected COS-7 cells with pEYFP plasmid.</p

Tnni3k protein levels.

<p>Immunoblot of atrial (A) and ventricular (V) protein lysates of AKR/J and DBA/2J mice using α-mTnni3k and α-ILK as loading control.</p

<i>In silico</i> and <i>in vivo</i> correlation of <i>Tnni3k</i> levels and PR interval duration.

<p>(A) <i>In silico</i> analysis: mouse inbred lines are colored based on the haplotypes in the minimal region of overlap; PR interval duration obtained from the mouse phenome database (<a href="http://phenome.jax.org/" target="_blank">http://phenome.jax.org/</a>) FVBN/J (blue) (low expression) and green (high <i>Tnni3k</i> expression), inbred lines with the reddish colors carry rs49812611 (associated with nonsense mediated decay). (B) <i>In vivo</i> analysis of <i>Tnni3k</i> expression levels (y-axis) and PR interval duration (x-axis) in six inbred mouse lines, colors again denote the haplotype (gray is unknown haplotype).</p

Genotype effects at rs13477506.

<p>(A) <i>Tnni3k</i> expression (Illumina probe ILMN_3023962) as a function of genotype at rs13477506 in F2 mice; homozygous 129P2: AA, green; 129P2-FVBN/J heterozygous: AB, turquoise; homozygous FVBN/J: BB, blue; the darker shades represent the independent validation of the <i>Tnni3k</i> transcription levels by Q-PCR (right y-axis). (B) PR interval as a function of genotype at rs13477506 in F2 mice. Error bars indicate standard errors.</p