Analysis Of Recombinant Inbred Strains Research Articles

Identification of the UBP1 Locus as a Critical Blood Pressure Determinant Using a Combination of Mouse and Human Genetics

Hypertension is a major health problem of largely unknown genetic origins. To identify new genes responsible for hypertension, genetic analysis of recombinant inbred strains of mice followed by human association studies might prove powerful and was exploited in our current study. Using a set of 27 recombinant BXD strains of mice we identified a quantitative trait locus (QTL) for blood pressure (BP) on distal chromosome 9. The association analysis of markers encompassing the syntenic region on human chromosome 3 gave in an additive genetic model the strongest association for rs17030583 C/T and rs2291897 G/A, located within the UBP1 locus, with systolic and diastolic BP (rs17030583: 1.3±0.4 mmHg p<0.001, 0.8±0.3 mmHg p = 0.006, respectively and rs2291897: 1.5±0.4 mmHg p<0.001, 0.8±0.3 mmHg p = 0.003, respectively) in three separate studies. Our study, which underscores the marked complementarities of mouse and human genetic approaches, identifies the UBP1 locus as a critical blood pressure determinant. UBP1 plays a role in cholesterol and steroid metabolism via the transcriptional activation of CYP11A, the rate-limiting enzyme in pregnenolone and aldosterone biosynthesis. We suggest that UBP1 and its functional partners are components of a network controlling blood pressure.

The Syntaxin Binding Protein 1 Gene (Stxbp1 ) Is a Candidate for an Ethanol Preference Drinking Locus on Mouse Chromosome 2

We previously mapped a quantitative trait locus (QTL) for ethanol preference drinking to mouse chromosome 2 (mapped with high confidence, LOD = 15.5, p = 3 x 10(-16)). The specific gene(s) in the QTL interval responsible for phenotypic variation in ethanol preference drinking has not been identified. In the current study, we investigated the association of the syntaxin binding protein 1 gene (Stxbp1) with ethanol preference drinking and other ethanol traits using a panel of B6 x D2 (BXD) recombinant inbred (RI) strains derived from the C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains. Confirmation analyses for ethanol consumption and withdrawal were performed using a large B6D2 F2 cross, short-term selected lines derived from the B6 and D2 progenitor strains, and standard inbred strains. BXD RI strain analysis detected provisional associations between Stxbp1 molecular variants and ethanol consumption, as well as severity of acute ethanol withdrawal, ethanol-conditioned taste aversion, and ethanol-induced hypothermia. Confirmation analyses using three independent genetic models supported the involvement of Stxbp1 in ethanol preference drinking but not in ethanol withdrawal. Stxbp1 encodes a Sec1/Munc18-type protein essential for vesicular neurotransmitter release. The present study provides supporting evidence for the involvement of Stxbp1 in ethanol preference drinking.

Genetic characterization of the Dyscalc locus.

Calcification occurs frequently in the development of atherosclerotic lesions, and studies in mice have indicated a genetic contribution. We now show that one genetic factor contributing to aortic calcification is the Dyscalc locus, previously shown to contribute to myocardial calcification. Thus, the Dyscalc locus, on proximal mouse Chromosome (Chr) 7, segregated with vascular calcification in a large cross between susceptible strain DBA/2J and resistant strain C57BL/6J. Further evidence was observed by analysis of recombinant inbred strains derived from various susceptible and resistant parental strains. Myocardial and vascular calcifications are importantly influenced by multiple modifier loci as well as the Dyscalc gene, making fine mapping of Dyscalc difficult. In order to allow more detailed genetic and biochemical characterization of Dyscalc, we have identified congenic strains containing the Dyscalc locus from resistant strain C57BL/10 on the background of susceptible strain C3H/DiSnA. The congenic strains exhibit little or no myocardial or vascular calcification, unlike the background HcB C3H strain, and the calcification segregated as a Mendelian factor, allowing finer mapping of Dyscalc.

The use of restriction landmark genomic scanning to scan the mouse genome for endogenous loci with imprinted patterns of methylation.

Restriction landmark genomic scanning (RLGS) has been used to screen endogenous loci for imprinted patterns of methylation. The screening method is based upon the identification of genetic variation in RLGS profiles between different strains and determining whether specific variant landmarks are transmitted equally to the progeny of reciprocal F1 matings. The RLGS profiles of C57BL/6 (B6) and DBA/2 (D2) and their reciprocal hybrids were produced with two enzyme combinations that used NotI as the landmark enzyme and two combinations that used BssHII. An estimated 13% of the spots are either B5- or D2-specific in these tests, giving a total of nearly 1000 variant loci that were examined for imprinted methylation. Three candidate loci for imprinted regulation were identified in these analyses. We also used crosses of more genetically diverse parents to increase the number of variant loci screened. Interspecific crosses of B6 with the M. musculus strain PWK and intrasubspecific crosses between B6 and the M. molossinus strain MSM expanded the levels of variation between the parental strains in the cross to an estimated 31% and 26%, respectively. The RLGS patterns for one NotI combination and one BssHII profile were examined for each of these crosses, giving approximately 2000 additional loci that were screened for imprinted patterns of methylation. Eight loci with imprinted patterns of transmission were observed out of 3040 loci tested. The chromosomal locations for the three B6 and D2 specific loci, Irlgs 1-3, were identified using BXD recombinant inbred strain analysis. Irlgs 1 and 3 are B6- and D2-specific loci that had the same strain distribution pattern which mapped to the central region of chromosome 9.(ABSTRACT TRUNCATED AT 250 WORDS)

USE OF RECOMBINANT INBRED STRAINS FOR EVALUATION OF INTERMEDIATE PHENOTYPES IN SPONTANEOUS HYPERTENSION

1. The HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway (BN.1x) rat, represent a very useful system for gene mapping and for genetic analysis of certain model diseases, such as spontaneous hypertension. 2. These RI strains were genotyped in multiple genetic polymorphisms and characterized in blood pressure and some intermediate phenotypes. 3. The analysis of RI strains has revealed that (i) a gene in the vicinity of the major histocompatibility complex (RT1) on chromosome 20, a kallikrein-related gene on chromosome 4 and the renin gene on chromosome 13 were significantly associated with blood pressure, and (ii) Na+ leak in red blood cells correlated with blood pressure whereas relative heart and kidney weights as well as platelet aggregation did not.

Studies of axonal regeneration in C57BL/6J and A/J mice

The genetic and biological nature of a deficiency in axonal regeneration in C57BL/6J mice was investigated. From analysis of recombinant inbred strains, the genetic basis for the deficient regeneration behaviours of C57BL/6J and A/J mice is deduced to involve multiple loci. The defect in axonal regeneration in C57BL/6J mice appears to be a delay rather than permanant impairment and appears to involve sensory more than motor axons.

Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.

Mapping genes in the mouse using single-strand conformation polymorphism analysis of recombinant inbred strains and interspecific crosses.

We have utilized a PCR-based analysis of single-strand conformation polymorphisms to identify polymorphisms that can be used for mapping cloned DNA sequences in the mouse. We have found that single-strand conformation polymorphism analysis of sequences that are potentially less subject to conservation (i.e., intron and 3' untranslated regions) is a relatively efficient means of detecting polymorphisms between inbred strains. Fifty percent of the tested primer pairs were polymorphic between inbred strains and 90% were polymorphic between mouse species, which is a frequency comparable to that found for microsatellite repeat sequences. We have found that this technique can be readily used to determine the strain distribution pattern in a recombinant inbred series and is a simple and rapid means to obtain a map position for cloned sequences. When this strategy was tested on a number of previously mapped cloned genes, the strain distribution patterns obtained were consistent with that to be expected on the basis of the known map position. We also tested the utility of this approach for characterizing genes that have not been previously mapped. Dvl, the mouse homolog of the putative Drosophila dishevelled gene, and Adfp, encoding an adipocyte differentiation-related protein, were found to map to chromosome 4. These results were confirmed using single-strand conformation polymorphism analysis of an interspecific backcross.

A recombinant inbred strain analysis of sleep-time responses to several sedative-hypnotics.

The results of previous studies suggested that the sleep-time differential between long-sleep (LS) and short-sleep (SS) mice decreases for a series of sedative-hypnotic drugs as lipid solubility increases. Using the LS x SS recombinant inbred (RI) strains, we have tested whether this relationship arises because of common genetic influences on the sleep-time responses or was simply a fortuitous difference between LS and SS mice. Mice were sleep-time tested after intraperitoneal injections of a variety of sedative-hypnotic drugs that vary in lipid solubility including alcohols, barbiturates, chloral hydrate, and urethane. Sleep-time values from each of these drugs were compared with ethanol-induced sleep times in the LS x SS RI strains. Significant genetic correlations were observed between ethanol-induced sleep time and those responses elicited by butanol, propanol, and chloral hydrate, but not by pentobarbital or secobarbital. When the partition coefficient (log P) values were compared with the genetic correlations, a significant relationship (r = -0.85) was observed. These data suggest that common genes mediate sedative-hypnotic reactions to ethanol and some drugs resembling it in log P value, and that anesthetic agents with low lipid solubility may be working through different mechanisms than drugs with greater lipid solubilities.

Single-locus control of saccharin intake in BXD/Ty recombinant inbred (RI) mice: some methodological implications for RI strain analysis.

The sac locus, with a major effect on saccharin preference, was discovered by Fuller (1974) in C57BL/6J (B6), DBA/2J (D2), and derived crosses, and is now supported in the BXD/Ty recombinant inbred (RI) series by a marked bimodal distribution in saccharin preference among 20 strains. The B6 allele led to increased saccharin preference compared to the D2 allele. Since the search for bimodal distributions reflecting major gene loci is an essential part of RI strain analysis, a new statistical method is proposed to test for bimodality, and comparisons are made to previously proposed methods. Another new RI method, quantitative trait loci (QTL) analysis, allows provisional detection and mapping of minor as well as major gene loci. Using this method as a screen, significant associations with saccharin preference were suggested with marker loci on portions of six chromosomes. One of these, the D12nyu1 locus on chromosome 12, was independently supported in a panel of standard (non-RI) inbred strains also tested for saccharin preference. It is unclear whether this reflects the sac locus.

Close linkage of retinoic acid receptor genes with homeobox- and keratin-encoding genes on paralogous segments of mouse chromosomes 11 and 15.

Retinoic acid is essential for normal development and growth of structures such as head and limbs, and it can act as morphogen or teratogen. Retinoic acid induces expression of genes such as the homeobox genes and keratin type I and type II genes. Retinoic acid receptors are nuclear transcription factors that play a key role in retinoid physiology. As part of the characterization of retinoic acid receptor gene family, linkage of genes encoding the three receptors was determined by using interspecific backcross and recombinant inbred strain analysis of restriction fragment variants. Retinoic acid receptor alpha is located on mouse Chromosome (Chr) 11 near the homeobox-2 complex and the keratin type I gene complex, whereas retinoic acid receptor gamma is on mouse Chr 15 near the homeobox-3 complex and the keratin type II complex. Close genetic proximity of these functionally related genes may be significant. We confirmed assignment of retinoic acid receptor beta to the centromeric portion of Chr 14. These linkage assignments provide further evidence for duplicated segments in the mouse genome.

A Congenital Interstitial Cell Hamartoma of the Equine Ovary

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Identification of a locus on mouse chromosome 3 involved in differential susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease

Theiler's virus-induced demyelinating disease results from a chronic infection in the white matter of the central nervous system and provides an excellent model for human multiple sclerosis. Like multiple sclerosis, there are genetic risk factors in disease development, including genes associated with the major histocompatibility complex and with those encoding the beta chain of the T-cell receptor. Comparisons of the susceptible DBA/2 and resistant C57BL/6 strains have indicated an important role for the H-2D locus and for a non-H-2 gene (not involving the beta chain of the T-cell receptor) in differential susceptibility. In the present report, analysis of recombinant-inbred strains (BXD) between the DBA/2 and C57BL/6 strains indicated that this non-H-2 locus is located at the centromeric end of chromosome 3 near (4 +/- 4 centimorgans) the carbonic anhydrase-2 (Car-2) enzyme locus.

Linkage of the mouse LDL receptor gene on chromosome 9

We identified restriction fragment length variants of the mouse low-density lipoprotein receptor gene and used these to map the gene, designated Ldlr, to the proximal region of chromosome 9. An interspecific backcross between strains MEV and CAST Ei yielded the following gene order and distances in centimorgans: Ldlr-18.8±5.6- Apoa-4-7.3±3.5- Mpi-1-10.2±3.9- Emv-3 or dilute. Analysis of recombinant inbred strains also indicated that Ldlr is tightly linked to a previously unmapped retroviral marker, Xmmv-67.

An analysis of spontaneous hypertension in spontaneously hypertensive rats by means of new recombinant inbred strains

The mode of blood pressure inheritance and some genetic markers of spontaneous hypertension were evaluated in a new set of recombinant inbred (RI) strains obtained by crossing of normotensive (BN.lx) and hypertensive (SHR) progenitor strains. Blood pressure values of RI strains were continuously distributed between both progenitor strains, although normotensive strains slightly prevailed. Statistical analysis suggested that there are three major genes and multiple minor genes responsible of the determination of spontaneous hypertension. The association between blood pressure and gene(s) within RT1 complex or gene(s) closely linked to it was found by RI strain analysis. This suggestion was confirmed by the detection of significant difference in blood pressure between SHR and SHR.1N congenic strains. Our results indicate that RT1 complex gene(s) may be involved in the development of high blood pressure.

Chromosomal organization of the cytochrome P450-2C gene family in the mouse: a locus associated with constitutive aryl hydrocarbon hydroxylase.

Cytochromes P-450 represent a superfamily of enzymes with a central role in the metabolism of drugs, chemical toxins, and carcinogens. We have used genetic analysis to establish the complexity and catalytic function of a recently identified constitutively expressed murine hepatic cytochrome P-450 encoded by P450-2C. Southern blotting analysis shows that there are at least seven or eight genes within this family in the mouse and rat and that DNA restriction fragment length variants between different mouse inbred strains are observed. Analysis of recombinant inbred strains derived from these parent strains shows (i) these genes are clustered within 1 centimorgan, (ii) this gene family does not correspond to any of the known cytochrome P-450 loci or map near any well-characterized genomic markers, and (iii) this gene family segregates to within 1-2 centimorgans of a locus controlling constitutive aryl hydrocarbon hydroxylase activity in mice. With use of Chinese hamster/mouse somatic cell hybrids, the P450-2C locus was assigned to a region of mouse chromosome 19 that appears to be syntenic with the previously mapped human P450C2C locus on human chromosome 10. By in situ hybridization to mitotic mouse chromosomes, we have localized this region to the tip of chromosome 19. These results are discussed in relation to the physiological roles of this P-450 family in foreign compound metabolism and steroid oxidations.

Major genes control hormone-induced ovulation rate in mice.

The present study examined the magnitude of genetic variation, mode of inheritance and number of loci controlling major genetic differences in hormone-induced ovulation rate in mice. Mice were injected with 5 i.u. PMSG at 28 days of age and 5 i.u. hCG at 30 days, and hormone-induced ovulation rate was determined from counts of oviducal eggs in cumulus the next morning. Six-fold genetic differences in induced ovulation rate were detected amongst strains, ranging from a low mean (+/- s.e.) value of 8.8 (+/- 0.9) in A/J up to 53.5 (+/- 2.2) in C57BL/6J. The number of ova differed significantly amongst strains and amongst F1 crosses (P less than 0.0001): 70% of the variation in hormone-induced ovulation rate was amongst strains. Of 9 F1 crosses examined, 4 showed positive heterosis, 3 showed no heterosis and 2 showed negative heterosis for hormone-induced ovulation rate. Analysis of parental, F1 and F2 generations revealed that the induced ovulation rate of A/J and C57BL/6J mice differed due to the action of about 3 or 4 loci, and A.SW/SnJ and SJL/J mice differed due to the action of about 2 to 3 loci. Analysis of recombinant inbred strains formed from A/J and C57BL/6J confirmed that these strains differed due to the action of a small number of loci. This study demonstrates the existence of a small number of major genes controlling hormone-induced ovulation rate in young mice.

Changes in size and cell populations in lymphoid organs during Plasmodium chabaudi infection in resistant and susceptible mice.

The level of host resistance to Plasmodium chabaudi is genetically determined by a major, dominant, autosomal, non H-2 linked gene which has been designated Pchr (for resistance to P. chabaudi) The genetic control was defined by our laboratory by analysis of hybrid and backcross progeny derived from the strain combination of resistant C57BL/6J (B) and susceptible A/J (A) mice and was confirmed by analysis of the AXB/BXA series of recombinant inbred (RI) strains (1,2). The RI strain analysis showed, furthermore, that resistance and, consequently, survival are genetically linked with marked splenomegaly while susceptibility and death are linked with minimal splenomegaly.

Genetic analysis of the proximal portion of the mouse t complex: Evidence for a second inversion within t haplotypes

Genomic sequences derived from the mouse t complex by a microdissection cloning technique have been used as tools to obtain high resolution genetic maps of the wild-type and t haplotype forms of the most proximal portion of chromosome 17. Genetic mapping was performed through a recombinant inbred strain analysis and an analysis of partial t haplotypes. The accumulated data demonstrate the existence of a large inversion of genetic material, encompassing the loci of T and qk, within the proximal portion of t haplotypes. This newly described proximal inversion and the previously described distal inversion provide an explanation for the suppression of recombination observed along the length of t haplotype DNA in heterozygous mice.

Confidence limits for estimates of gene linkage based on analysis of recombinant inbred strains.

Recombinant inbred (RI) strains are extremely useful for genetic mapping. This paper presents a simple method for determining confidence intervals for linkage estimates based on analysis of RI strains. The results show that such confidence intervals are usually large with the currently available numbers of RI strains. Therefore, map positions based only on analysis of RI strains should be interpreted with caution. To facilitate interpretation of linkage data derived from RI strains, a table is presented giving the 95 percent and 99 percent confidence intervals for all possible linkages detected with up to 45 RI strains.