Identification of Stim1 as a Candidate Gene for Exaggerated Sympathetic Response to Stress in the Stroke-Prone Spontaneously Hypertensive Rat

The stroke-prone spontaneously hypertensive rat (SHRSP) is known to have exaggerated sympathetic nerve activity to various types of stress, which might contribute to the pathogenesis of severe hypertension and stroke observed in this strain. Previously, by using a congenic strain (called SPwch1.72) constructed between SHRSP and the normotensive Wistar-Kyoto rat (WKY), we showed that a 1.8-Mbp fragment on chromosome 1 (Chr1) of SHRSP harbored the responsible gene(s) for the exaggerated sympathetic response to stress. To further narrow down the candidate region, in this study, another congenic strain (SPwch1.71) harboring a smaller fragment on Chr1 including two functional candidate genes, Phox2a and Ship2, was generated. Sympathetic response to cold and restraint stress was compared among SHRSP, SPwch1.71, SPwch1.72 and WKY by three different methods (urinary norepinephrine excretion, blood pressure measurement by the telemetry system and the power spectral analysis on heart rate variability). The results indicated that the response in SPwch1.71 did not significantly differ from that in SHRSP, excluding Phox2a and Ship2 from the candidate genes. As the stress response in SPwch1.72 was significantly less than that in SHRSP, it was concluded that the 1.2-Mbp congenic region covered by SPwch1.72 (and not by SPwch1.71) was responsible for the sympathetic stress response. The sequence analysis of 12 potential candidate genes in this region in WKY/Izm and SHRSP/Izm identified a nonsense mutation in the stromal interaction molecule 1 (Stim1) gene of SHRSP/Izm which was shared among 4 substrains of SHRSP. A western blot analysis confirmed a truncated form of STIM1 in SHRSP/Izm. In addition, the analysis revealed that the protein level of STIM1 in the brainstem of SHRSP/Izm was significantly lower when compared with WKY/Izm. Our results suggested that Stim1 is a strong candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP.     

Blood Pressure-Independent Factors Determine the Susceptibility to Delayed Neuronal Death in the Stroke-Prone Spontaneously Hypertensive Rats

The stroke-prone spontaneously hypertensive rat (SHRSP) is vulnerable to delayed neuronal death (DND) in the CA1 subfield of the hippocampus after the transient forebrain ischemia by the occlusion of the bilateral carotid arteries. The present study was designed to show that the genetic factors independent of high blood pressure contributed to the high incidence of DND in SHRSP. Male rats of the four strains, SHRSP/Izm, SHRSP/Ngsk, SHR/Izm and a congenic strain for the blood pressure quantitative trait locus on chromosome 1 [SHRSP.WKY-(D1Wox29-D1Arb21)/Izm]were used in the experiments. At 13 weeks of age, the bilateral carotid arteries of rats were occluded for 10 min under anesthesia with their body temperature kept at 37°C. Seven days after the transient ischemia, the loss of the pyramidal cells in the CA1 was evaluated histologically. In some experiments, the blood flow was monitored with a laser Doppler flowmeter during the transient ischemia. The blood pressure in SHRSP/Izm was significantly greater than that in the other three strains. The incidence of DND, however, was not significantly different among SHRSP/Izm, SHRSP/Ngsk and the congenic strain (82, 74 and 65%, respectively), while SHR/Izm showed a significantly lower incidence (20%). Neither a significant correlation between the incidence of DND and the blood flow reduction during the occlusion, nor a significant inter-strain difference in the blood flow reduction was observed. The genetic factors independent of high blood pressure may contribute to the greater susceptibility to DND in SHRSP.     

Characterizing Photothrombotic Distal Middle Cerebral Artery Occlusion and YAG Laser-Induced Reperfusion Model in the Izumo Strain of Spontaneously Hypertensive Rats

No study has systematically studied the relevance of original Izumo strain of spontaneously hypertensive rats (SHR/Izm) as a stroke model. Furthermore, both SHR/Izm and stroke-prone SHR/Izm (SHRSP/Izm) are commercially available, and recent progress in genetic studies allowed us to use several congenic strains of rats constructed with SHR/Izm and SHRSP/Izm as the genetic background strains. A total of 166 male SHR/Izm and 17 male SHRSP/Izm were subjected to photothrombotic middle cerebral artery (MCA) occlusion with or without YAG laser-induced reperfusion. The pattern of distal MCA was recorded. Infarct volumes were determined with 2,3,5-triphenyltetrazolium chloride. At 24 or 48 h after MCA occlusion, infarct volumes in the permanent occlusion and 2-h occlusion groups (88 ± 22 [SD] and 87 ± 25 mm³, respectively) were significantly larger than that in the 1-h occlusion group (45 ± 14 mm³), indicating the presence of sizeable zone of penumbra. Infarct size in SHRSP/Izm determined at 24 h after MCA occlusion was fairly large (124.0 ± 34.8 mm³, n = 10). Infarct volume in SHR/Izm with simple distal MCA was 76 ± 19 mm³, which was significantly smaller than 95 ± 22 mm³ in the other SHR/Izm with more branching MCA. These data suggest that this stroke model in SHR/Izm is useful in the preclinical testing of stroke therapies and elucidating the pathophysiology of cerebral ischemia/reperfusion.     

Pathological alterations of astrocytes in stroke-prone spontaneously hypertensive rats under ischemic conditions

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension, and more than 95% of them die of cerebral stroke. We showed the vulnerability of neuronal cells of SHRSP/Izm rats. Furthermore, we analyzed the characteristics of SHRSP/Izm astrocytes during a stroke. It is known that the proliferating ability of SHRSP/Izm astrocytes is significantly enhanced compared with those in the normotensive Wistar Kyoto rats (WKY/Izm) strain. Conversely, the ability of SHRSP/Izm astrocytes to form tight junctions (TJ) was attenuated compared with astrocytes from WKY/Izm rats. During the stress of hypoxia and reoxygenation (H/R), lactate production, an energy source for neuronal cells, decreased in SHRSP/Izm astrocytes in comparison with the WKY/Izm strain. Moreover, during H/R, SHRSP/Izm astrocytes decreased their production of glial cell line-derived neurotrophic factor (GDNF) in comparison with WKY/Izm astrocytes. Furthermore, SHRSP/Izm rats decreased production of l-serine, compared with WKY/Izm rats following nitric oxide (NO) stimulation. Additionally, in H/R, astrocytes of SHRSP/Izm rats expressed adhesion molecules such as VCAM-1 at higher levels.It is possible that all of these differences between SHRSP/Izm and WKY/Izm astrocytes are not associated with the neurological disorders in SHRSP/Izm. However, attenuated production of lactate and reduced GDNF production in astrocytes may reduce required energy levels and weaken the nutritional status of SHRSP/Ism neuronal cells. We suggest that the attenuation of astrocytes’ functions accelerates neuronal cell death during stroke, and may contribute to the development of strokes in SHRSP/Izm. In this review, we summarize the altered properties of SHRSP/Izm astrocytes during a stroke.     

Dietary β-carotene regulates interleukin-1β-induced expression of apolipoprotein E in astrocytes isolated from stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) have an abnormality in cholesterol synthesis, but the pathological relevance of this to stroke and related neuronal disorders is not yet clear. The induction of astrocyte-derived cholesterol transportation to neurons by apolipoprotein E (apoE) promotes neuronal repair after brain injuries such as stroke. Such repair is reduced by interleukin-1 beta (IL-1β) and stroke conditions. Furthermore, fibroblast growth factor 1 (FGF1) regulates the production of apoE–cholesterol-rich high density lipoproteins (HDL) and induces gliosis of astrocytes. On the other hand, high levels of plasma carotenoids reduce the risk of ischemic stroke. Thus, we investigated the expression of apoE in primary astrocytes that had been treated with IL-1β or β-carotene. In addition, we compared the expression levels of Apoe genes in astrocytes from SHRSP/Izm and normal control rats, Wistar–Kyoto rats (WKY/Izm) following hypoxia/reoxygenation (H/R). The expression levels of genes and proteins were investigated by RT-PCR, Western blotting (WB), and immunofluorescence analysis. IL-1β decreased the expression levels of the Apoe gene. Conversely, β-carotene significantly enhanced the expression levels of genes related to cholesterol regulation, including Abca1, Abcg1, Hmgcr as well as Apoe. During H/R, the gene expression levels of Apoe were decreased in the SHRSP/Izm rats in comparison with the WKY/Izm rats. These results suggest that IL-1β decreases Apoe expression levels, whereas β-carotene strongly elevates Apoe levels and inhibits FGF1-mediated gliosis of astrocytes. Furthermore, under hypoxic stress, astrocytes isolated from SHRSP/Izm rats displayed altered regulation of Apoe compared with those from WKY/Izm rats.     

SHRSP/Izm and WKY/NCrlCrlj Rats Having a Missense Mutation in Abcg5 Deposited Plant Sterols in the Body,but Did Not Change Their Biliary Secretion and Lymphatic Absorption—Comparison with Jcl:Wistar and WKY/Izm Rats

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.     

Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.     

Nutritional Prevention on Hypertension, Cerebral Hemodynamics and Thrombosis in Stroke-Prone Spontaneously Hypertensive Rats

1. Stroke-prone spontaneously hypertensive rats (SHRSP/Izm), which become severely hypertensive and exhibit a very high incidence of stroke (cerebral hemorrhage and/or infarction), are used widely for the study of the hypertension and stroke. In the previous study, we indicated that high thrombotic tendency of cerebral microvessels in SHRSP/Izm compared with stroke-resistant SHR (SHR/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) at aged period. 2. L-arginine, a substrate of nitric oxide (NO), and voluntary exercise reduced blood pressure and thrombotic tendency in cerebral microvessels caused by highly production of NO in vivo. Furthermore, antioxidants show that the effects of antihypertensive and antithrombosis in SHRSP/Izm. 3. Although SHRSP/Izm become genetically hypertensive and exhibit stroke, a number of nutritional factors, particularly antioxydative nutrient, have preventive effects on hypertension, cerebral blood flow dysfunction, thrombus formation, and neuronal cell death in SHRSP/Izm. Our results indicate that those treatments are beneficial in the prevention of hypertension and stroke and that the nutritional science is very important for "prediction and prevention medicine."     

Genetic analysis of testis weight and fertility in an interspecies hybrid congenic strain for Chromosome X

A hybrid congenic strain, C57BL/6J.SPRET-Hprt ^a , carrying 17 map units of Chromosome (Chr) X from Mus spretus on a background of C57BL/6J, has the novel phenotype of low fertility associated with small testis weight. In histological cross-section, many of the tubules in the testes of these congenic mice are empty except for Sertoli cells, while the other tubules appear to be normal. The gene, interspecific hybrid testis weight 1 (Ihtw1) causing this phenotype, has been fine mapped by using the strategy of generating subcongenic strains from recombinants within the congenic region. Genetic and phenotypic analysis of the subcongenic strains has defined a critical region of 1.8 map units for Ihtw1. This region of the genetic map is orthologous to the region on human Chr X containing the gene for the Borjeson-Forssman-Lehman syndrome, an inherited disease in which males show microorchidism. Received: 12 June 2000 / Accepted: 8 September 2000     

Intersubspecific subcongenic mouse strain analysis reveals closely linked QTLs with opposite effects on body weight

A previous genome-wide QTL study revealed many QTLs affecting postnatal body weight and growth in an intersubspecific backcross mouse population between the C57BL/6J (B6) strain and wild Mus musculus castaneus mice captured in the Philippines. Subsequently, several closely linked QTLs for body composition traits were revealed in an F₂ intercross population between B6 and B6.Cg-Pbwg1, a congenic strain on the B6 genetic background carrying the growth QTL Pbwg1 on proximal chromosome 2. However, no QTL affecting body weight has been duplicated in the F₂ population, except for mapping an overdominant QTL that causes heterosis of body weight. In this study, we developed 17 intersubspecific subcongenic strains with overlapping and nonoverlapping castaneus regions from the B6.Cg-Pbwg1 congenic strain in order to search for and genetically dissect QTLs affecting body weight into distinct closely linked loci. Phenotypic comparisons of several developed subcongenic strains with the B6 strain revealed that two closely linked but distinct QTLs that regulate body weight, named Pbwg1.11 and Pbwg1.12, are located on an 8.9-Mb region between D2Mit270 and D2Mit472 and on the next 3.6-Mb region between D2Mit205 and D2Mit182, respectively. Further analyses using F₂ segregating populations obtained from intercrosses between B6 and each of the two selected subcongenic strains confirmed the presence of these two body weight QTLs. Pbwg1.11 had an additive effect on body weight at 6, 10, and 13?weeks of age, and its castaneus allele decreased it. In contrast, the castaneus allele at Pbwg1.12 acted in a dominant fashion and surprisingly increased body weight at 6, 10, and 13?weeks of age despite the body weight of wild castaneus mice being 60% of that of B6 mice. These findings illustrate the complex genetic nature of body weight regulation and support the importance of subcongenic mouse analysis to dissect closely linked loci.     

Increased physical activity cosegregates with higher intake of carbohydrate and total calories in a subcongenic mouse strain

C57BL/6 J (B6) and CAST/EiJ (CAST), the inbred strain derived from M. musculus castaneus, differ in nutrient intake behaviors, including dietary fat and carbohydrate consumption in a two-diet-choice paradigm. Significant quantitative trait loci (QTLs) for carbohydrate (Mnic1) and total energy intake (Kcal2) are present between these strains on chromosome (Chr) 17. Here we report the refinement of the Chr 17 QTL in a subcongenic strain of the B6.CAST- ^{D17Mit19-D17Mit91} congenic mice described previously. This new subcongenic strain possesses CAST Chr 17 donor alleles from 4.8 to 45.4 Mb on a B6 background. Similar to CAST, the subcongenic mice exhibit increased carbohydrate and total calorie intake per body weight, while fat intake remains equivalent. Unexpectedly, this CAST genomic segment also confers two new physical activity phenotypes: 22% higher spontaneous physical activity levels and significantly increased voluntary wheel-running activity compared with the parental B6 strain. Overall, these data suggest that gene(s) involved in carbohydrate preference and increased physical activity are contained within the proximal region of Chr 17. Interval-specific microarray analysis in hypothalamus and skeletal muscle revealed differentially expressed genes within the subcongenic region, including neuropeptide W (Npw); glyoxalase I (Glo1); cytochrome P450, family 4, subfamily f, polypeptide 1 (Cyp4f15); phospholipase A2, group VII (Pla2g7); and phosphodiesterase 9a (Pde9a). This subcongenic strain offers a unique model for dissecting the contributions and possible interactions among genes controlling food intake and physical activity, key components of energy balance.     

Identification of a congenic mouse line with obesity and body length phenotypes

Our primary objective was to discover simplified mouse models corresponding to human obesity linkages. We used the B10.UW– H3^b we Pax1^un a^t/Sn (B10.UW) congenic strain, a subcongenic strain with a reduced UW strain donor region, and their C57BL/10SnJ background strain. The congenic and subcongenic UW strain donor regions are on mouse Chr 2. We measured body length [anal-nasal (AN) length], summed fat depot weights normalized for body weight (Adiposity Index, AI), and percentage of body weight that is lipid. The B10.UW congenic and subcongenic strains have significantly smaller AN lengths (p < 0.0001) and have a significantly lower AI and percentage of body weight as fat than the background strain (p < 0.0001). In an F₂ intercross of the congenic and background strains, AN and AI were both linked to the distal half of the donor region with LOD scores greater than 19 and 5, respectively. F₂ haplotypes identified a minimal region for AN linkage of 0.8 megabases (Mb) that is estimated to express four genes in the current Celera mouse genome assembly. We narrowed the most likely location of the obesity gene to 15 Mb whose homologous genes are all located on human Chr 20 in the region surrounding the centromere. Since a previous study identified human obesity linkage peaking near the centromere, then the B10.UW mice may exhibit obesity due to the homologous gene.     

Two new behavioral QTLs, Emo4 and Reb1, map to mouse Chromosome 1: Congenic strains and candidate gene identification studies

By use of newly developed subcongenic strains of mice from a parental B6.129-Il10^−/− knockout/congenic strain, we have narrowed the critical region for a new behavioral QTL, called Emo4, for open-field activity to a segment of Chromosome 1 between Erbb4 (68.4Mb) and B3gnt7 (86.2 Mb). We have also uncovered an additional QTL governing repetitive beam breaks in the open field. This QTL, called Reb1, maps to the interval between Asb1 (91.4 Mb) and NM_172851 (100.0 Mb) and is one of the first QTLs mapped for this type of behavior. Genome-wide microarray expression analyses were then undertaken to help to identify candidate genes that may be the cause of these genetic differences in open-field performance. In this effort, we analyzed global gene expression differences in the amygdalae by use of Affymetrix GeneChips between B6, B6.129-Il10^−/−, and B6.129R4. Several probe sets representing target Chr 1 genes were found that showed significantly differential expression in the subcongenic and congenic strains. Several candidate genes have been identified. One of these regions coincides with an homologous region in humans that has been associated with autism, a disease whose symptoms include repetitive actions. This study illustrates that the use of congenic strains combined with global gene expression analyses can produce a list of viable candidates. It further shows that caution should be observed when analyzing the effects of knockout/congenic strains because many of the gene expression differences in these comparisons could not be attributable to the ablated Il10 gene but rather to passenger gene effects.     

Overlapping mouse subcongenic strains successfully separate two linked body fat QTL on distal MMU 2

Background

Mouse chromosome 2 is linked to growth and body fat phenotypes in many mouse crosses. With the goal to identify the underlying genes regulating growth and body fat on mouse chromosome 2, we developed five overlapping subcongenic strains that contained CAST/EiJ donor regions in a C57BL/6J^hg/hg background (hg is a spontaneous deletion of 500 Kb on mouse chromosome 10). To fine map QTL on distal mouse chromosome 2 a total of 1,712 F2 mice from the five subcongenic strains, plus 278 F2 mice from the HG2D founder congenic strain were phenotyped and analyzed. Interval mapping (IM) and composite IM (CIM) were performed on body weight and body fat traits on a combination of SNP and microsatellite markers, which generated a high-density genotyping panel.

Results

Phenotypic analysis and interval mapping of total fat mass identified two QTL on distal mouse chromosome 2. One QTL between 150 and 161 Mb, Fatq2a, and the second between 173.3 and 175.6 Mb, Fatq2b. The two QTL reside in different congenic strains with significant total fat differences between homozygous cast/cast and b6/b6 littermates. Both of these QTL were previously identified only as a single QTL affecting body fat, Fatq2. Furthermore, through a novel approach referred here as replicated CIM, Fatq2b was mapped to the Gnas imprinted locus.

Conclusions

The integration of subcongenic strains, high-density genotyping, and CIM succesfully partitioned two previously linked QTL 20 Mb apart, and the strongest QTL, Fatq2b, was fine mapped to a ~2.3 Mb region interval encompassing the Gnas imprinted locus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1191-8) contains supplementary material, which is available to authorized users.     

Positional Cloning Reveals Strain-Dependent Expression of Trim16 to Alter Susceptibility to Bleomycin-Induced Pulmonary Fibrosis in Mice

Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named Blmpf1 and Blmpf2. In this study we isolated the quantitative trait gene underlying Blmpf2 initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, Trim16 was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of Trim16 by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H-Blmpf2 subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in Trim16 leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice.     

Narrowing a region on rat chromosome 13 that protects against hypertension in Dahl SS-13BN congenic strains

Transfer of chromosome 13 from the Brown Norway (BN) rat onto the Dahl salt-sensitive (SS) genetic background attenuates the development of hypertension, but the genes involved remain to be identified. The purpose of the present study was to confirm by telemetry that a congenic strain [SS.BN-(D13Hmgc37-D13Got22)/Mcwi, line 5], carrying a 13.4-Mb segment of BN chromosome 13 from position 32.4 to 45.8 Mb, is protected from the development of hypertension and then to narrow the region of interest by creating and phenotyping 11 additional subcongenic strains. Mean arterial pressure (MAP) rose from 118 ± 1 to 186 ± 5 mmHg in SS rats fed a high-salt diet (8.0% NaCl) for 3 wk. Protein excretion increased from 56 ± 11 to 365 ± 37 mg/day. In contrast, MAP only increased to 152 ± 9 mmHg in the line 5 congenic strain. Six subcongenic strains carrying segments of BN chromosome 13 from 32.4 and 38.2 Mb and from 39.9 to 45.8 Mb were not protected from the development of hypertension. In contrast, MAP was reduced by ～30 mmHg in five strains, carrying a 1.9-Mb common segment of BN chromosome 13 from 38.5 to 40.4 Mb. Proteinuria was reduced by ～50% in these strains. Sequencing studies did not identify any nonsynonymous single nucleotide polymorphisms in the coding region of the genes in this region. RT-PCR studies indicated that 4 of the 13 genes in this region were differentially expressed in the kidney of two subcongenic strains that were partially protected from hypertension vs. those that were not. These results narrow the region of interest on chromosome 13 from 13.4 Mb (159 genes) to a 1.9-Mb segment containing only 13 genes, of which 4 are differentially expressed in strains partially protected from the development of hypertension.     

SHRSP/Izm and WKY/NCrlCrlj rats having a missense mutation in Abcg5 deposited plant sterols in the body, but did not change their biliary secretion and lymphatic absorption-comparison with Jcl:Wistar and WKY/Izm rats

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.     

Congenic mapping of a blood pressure QTL region on rat chromosome 10 using the Dahl salt-sensitive rat with introgressed alleles from the Milan normotensive strain

Multiple blood pressure (BP) quantitative trait loci (QTLs) are reported on rat chromosome 10 (RNO10). Of these, QTLs detected by contrasting the genome of the hypertensive Dahl salt-sensitive (S) rat with two different relatively normotensive strains, Lewis (LEW) and the Milan normotensive strain (MNS), are reported. Because the deduced QTL regions of both S vs. LEW and S vs. MNS comparisons are within large genomic segments encompassing more than 2 cM, there was a need to further localize these QTLs and determine whether the QTLs are unique to specific strain comparisons. Previously, the S.MNS QTL1 was mapped to less than 2.6 cM as a differential segment between two congenic strains. In this study, multiple congenic strains spanning the projected interval were studied. The BP effect of each strain was interpreted as the net effect of alleles introgressed within that congenic strain. The results suggest that the MNS alleles within the previously proposed differential segment (D10Rat27-D10Rat24) do not independently lower BP of the S rat. However, another congenic strain, S.MNS(10) × 9, containing introgressed MNS alleles that are outside of the previously proposed differential segment is of interest because (1) it demonstrated a BP-lowering effect, (2) it is contained within a single congenic strain and is not based on the observed effect of a differential segment, and, more importantly, (3) it overlaps with the previously identified S.LEW BP QTL region. Identification of the same QTL affecting BP in multiple rat strains will provide further support for the QTL’s involvement and importance in human essential hypertension.     

A locus on mouse Ch10 influences susceptibility to limbic seizure severity: fine mapping and in silico candidate gene analysis

Identification of genes contributing to mouse seizure susceptibility can reveal novel genes or pathways that provide insight into human epilepsy. Using mouse chromosome substitution strains and interval‐specific congenic strains (ISCS), we previously identified an interval conferring pilocarpine‐induced limbic seizure susceptibility on distal mouse chromosome 10 (Ch10). We narrowed the region by generating subcongenics with smaller A/J Ch10 segments on a C57BL/6J (B6) background and tested them with pilocarpine. We also tested pilocarpine‐susceptible congenics for 6‐Hz ECT (electroconvulsive threshold), another model of limbic seizure susceptibility, to determine whether the susceptibility locus might have a broad effect on neuronal hyperexcitability across more than one mode of limbic seizure induction. The ISCS Line 1, which contained the distal 2.7 Mb segment from A/J (starting at rs29382217), was more susceptible to both pilocarpine and ECT. Line 2, which was a subcongenic of Line 1 (starting at rs13480828), was not susceptible, thus defining a 1.0 Mb critical region that was unique to Line 1. Bioinformatic approaches identified 45 human orthologs within the unique Line 1 susceptibility region, the majority syntenic to human Ch12. Applying an epilepsy network analysis of known and suspected excitability genes and examination of interstrain genomic and brain expression differences revealed novel candidates within the region. These include Stat2, which plays a role in hippocampal GABA receptor expression after status epilepticus, and novel candidates Pan2, Cdk2, Gls2 and Cs, which are involved in neural cell differentiation, cellular remodeling and embryonic development. Our strategy may facilitate discovery of novel human epilepsy genes .     

Fine map of the Gct1 spontaneous ovarian granulosa cell tumor locus

The spontaneous development of juvenile-onset, ovarian granulosa cell (GC) tumors in the SWR/Bm (SWR) inbred mouse strain is a model for juvenile-type GC tumors that appear in infants and young girls. GC tumor susceptibility is supported by multiple Granulosa cell tumor (Gct) loci, but the Gct1 locus on Chr 4 derived from SWR strain background is fundamental for GC tumor development and uniquely responsive to the androgenic precursor dehydroepiandrosterone (DHEA). To resolve the location of Gct1 independently from other susceptibility loci, Gct1 was isolated in a congenic strain that replaces the distal segment of Chr 4 in SWR mice with a 47 × 10⁶-bp genomic segment from the Castaneus/Ei (CAST) strain. SWR females homozygous for the CAST donor segment were confirmed to be resistant to DHEA- and testosterone-induced GC tumorigenesis, indicating successful exchange of CAST alleles (Gct1 ^CA ) for SWR alleles (Gct1 ^SW ) at this tumor susceptibility locus. A series of nested, overlapping, congenic sublines was created to fine-map Gct1 based on GC tumor susceptibility under the influence of pubertal DHEA treatment. Twelve informative lines have resolved the Gct1 locus to a 1.31 × 10⁶-bp interval on mouse Chr 4, a region orthologous to human Chr 1p36.22.