Limited WKY chromosomal regions confer increases in anxiety and fear memory in a F344 congenic rat strain.

This study investigated the interaction between genetic differences in stress reactivity/coping and environmental challenges, such as acute stress during adolescence on adult contextual fear memory and anxiety-like behaviors. Fischer 344 (F344) and the inbred F344;WKY-Stresp3/Eer congenic strain (congenic), in which chromosomal regions from the Wistar-Kyoto (WKY) strain were introgressed into the F344 background, were exposed to a modified forced swim test during adolescence, while controls were undisturbed. In adulthood, fear learning and memory, assessed by contextual fear conditioning, were significantly greater in congenic animals compared with F344 animals, and stress during adolescence increased them even further in males of both strains. Anxiety-like behavior, measured by the open field test, was also greater in congenic than F344 animals, and stress during adolescence increased it further in both strains of adult males. Whole genome sequencing of the F344;WKY-Stresp3/Eer strain revealed an enrichment of WKY genotypes in chromosomes 9, 14, and 15. An example of functional WKY sequence variations in the congenic strain, cannabinoid receptor interacting protein 1 (Cnrip1) had a Cnrip1 transcript isoform that lacked two exons. Although the original hypothesis that the genetic predisposition to increased anxiety of the WKY donor strain would exaggerate fear memory relative to the background strain was confirmed, the consequences of adolescent stress were strain independent but sex dependent in adulthood. Molecular genomic approaches combined with genetic mapping of WKY sequence variations in chromosomes 9, 14, and 15 could aid in finding quantitative trait genes contributing to the variation in fear memory.NEW & NOTEWORTHY This study found that 1) whole genome sequencing of congenic strains should be a criterion for their recognition; 2) sequence variations between Wistar-Kyoto and Fischer 344 strains at regions of chromosomes 9, 14, and 15 contribute to differences in contextual fear memory and anxiety-like behaviors; and 3) stress during adolescence affects these behaviors in males, but not females, and is independent of strain.

NOD alleles at Idd1 and Idd2 loci drive exocrine pancreatic inflammation.

Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes and have enabled the identification of several loci associated with diabetes susceptibility, termed insulin-dependent diabetes (Idd). The generation of congenic mice has allowed the characterization of the impact of several loci on disease susceptibility. For instance, NOD.B6-Idd1 and B6.NOD-Idd1 congenic mice were instrumental in demonstrating that susceptibility alleles at the MHC locus (known as Idd1) are necessary but not sufficient for autoimmune diabetes progression. We previously showed that diabetes resistance alleles at the Idd2 locus provide significant protection from autoimmune diabetes onset, second to Idd1. In search of the minimal genetic factors required for T1D onset, we generated B6.Idd1.Idd2 double-congenic mice. Although the combination of Idd1 and Idd2 is not sufficient to induce diabetes onset, we observed immune infiltration in the exocrine pancreas of B6.Idd2 mice, as well as an increase in neutrophils and pancreatic tissue fibrosis. In addition, we observed phenotypic differences in T-cell subsets from B6.Idd1.Idd2 mice relative to single-congenic mice, suggesting epistatic interaction between Idd1 and Idd2 in modulating T-cell function. Altogether, these data show that Idd1 and Idd2 susceptibility alleles are not sufficient for autoimmune diabetes but contribute to inflammation and immune infiltration in the pancreas.

Modified cell trace violet proliferation assay preserves lymphocyte viability and allows spectral flow cytometry analysis.

In this study we describe three different methods for labeling T lymphocytes with cell trace violet (CTV), in order to track cell division in mouse and human cells, in both the in vitro and in vivo setting. We identified a modified method of CTV labeling that can be applied directly to either conventional or spectral flow cytometry, that maintained lymphocyte viability and function, yet minimized dye spill-over into other fluorochrome channels. Our optimized method for CTV labeling allowed us to identify up to eight cell divisions and the replication index for in vitro-stimulated mouse and human lymphocytes, and the co-expression of T-cell subset markers. Furthermore, the homeostatic trafficking, expansion and division of CTV-labeled congenic donor T cells could be detected using spectral cytometry, in an adoptive T-cell transfer mouse model. Our optimized CTV method can be applied to both in vitro and in vivo settings to examine the behavior and phenotype of activated T cells.

Shifting Paradigm from Gene Expressions to Pathways Reveals Physiological Mechanisms in Blood Pressure Control in Causation.

Genetics for blood pressure (BP) in human and animals has been partitioned into two separate specialties. However, this divide is mechanistically-misleading. BP physiology is mechanistically participated by products of quantitative trait loci (QTLs). The key to unlocking its mechanistic mystery lies in the past with mammalian ancestors before humans existed. By pivoting from effects to causes, physiological mechanisms determining BP by six QTLs have been implicated. Our work relies on congenic knock-in genetics in vivo using rat models, and has reproduced the physiological outcome based on a QTL being molecularly equal to one gene. A gene dose for a QTL is irrelevant to physiological BP controls in causation. Together, QTLs join one another as a group in modularized Mendelian fashion to achieve polygenicity. Mechanistically, QTLs in the same module appear to function in a common pathway. Each is involved in a different step in the pathway toward polygenic hypertension. This work has implicated previously-concealed components of these pathways. This emerging concept is a departure from the human-centric precept that the level of QTL expressions, not physiology, would ultimately determine BP. The modularity/pathway paradigm breaks a unique conceptual ground for unravelling the physiological mechanisms of polygenic and quantitative traits like BP.

Protein digestibility and bioavailability in an F2 mouse cross between the selected fat mouse line and an M2 congenic line carrying the anti-obesity and anti-diabetic Tst allele.

The study was performed to investigate protein digestibility and utilization in an F2 cross (M2-F2 cross) between the selected Fat (F) line and an M2 congenic line. The congenic M2 line carried the Fob3b2 quantitative trait locus (QTL) from the selected Lean (L) line previously shown to contain the Tst gene with leanness, anti-diabetic and resistance to diet-induced obesity effects. The main objective of the study was to test if some of the effects on leanness and obesity resistance of the L-line Fob3b2 could also be due to the effect of this QTL on nutrient digestibility and bioavailability. The F2 littermates carrying either the Fat line segment within the Fob3b2 region or the L-line were compared when fed the high-fat diet. Eleven mice per genotype were individually housed in metabolic cages. In 5-day experimental period, body mass and diet intake were measured. The part of study was done on the F and L line and tested the difference in apparent protein digestibility on low-fat (LFD) and high-fat (HFD) diet. The nitrogen content was determined in the diet, faeces, and urine based on which, the apparent protein digestibility, apparent protein biological value and apparent net protein utilization were calculated There were no significant differences in any of these parameters on congenic line, confirming that the phenotypic effect on adiposity between the genotypes in the M2-F2 population was not due to the differential effect of the Fob3b2 locus carrying the Tst gene on protein utilization. We conclude that the observed phenotypic effects of this gene region are due to direct metabolic actions rather than the effects on nutrient absorption and nitrogen utilization since there were no differences in apparent protein digestibility between L and F lines, irrespective to HFD or LFD. The age of animals had significant effect on the level of digestibility.

Evidence of genetic epistasis in autoimmune diabetes susceptibility revealed by mouse congenic sublines.

Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4-CD8- double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2k-Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2k-Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility.

Novel enhanced GFP-positive congenic inbred strain establishment and application of tumor-bearing nude mouse model.

Transgenic GFP gene mice are widely used. Given the unique advantages of immunodeficient animals in the field of oncology research, we aim to establish a nude mouse inbred strain that stably expresses enhanced GFP (EGFP) for use in transplanted tumor microenvironment (TME) research. Female C57BL/6-Tg(CAG-EGFP) mice were backcrossed with male BALB/c nude mice for 11 generations. The genotype and phenotype of novel inbred strain Foxn1nu .B6-Tg(CAG-EGFP) were identified by biochemical loci detection, skin transplantation and flow cytometry. PCR and fluorescence spectrophotometry were performed to evaluate the relative expression of EGFP in different parts of the brain. Red fluorescence protein (RFP) gene was stably transfected into human glioma stem cells (GSC), SU3, which were then transplanted intracerebrally or ectopically into Foxn1nu .B6-Tg(CAG-EGFP) mice. Cell co-expression of EGFP and RFP in transplanted tissues was further analyzed with the Live Cell Imaging System (Cell'R, Olympus) and FISH. The inbred strain Foxn1nu .B6-Tg(CAG-EGFP) shows different levels of EGFP expression in brain tissue. The hematological and immune cells of the inbred strain mice were close to those of nude mice. EGFP was stably expressed in multiple sites of Foxn1nu .B6-Tg(CAG-EGFP) mice, including brain tissue. With the dual-fluorescence tracing transplanted tumor model, we found that SU3 induced host cell malignant transformation in TME, and tumor/host cell fusion. In conclusion, EGFP is differentially and widely expressed in brain tissue of Foxn1nu .B6-Tg(CAG-EGFP), which is an ideal model for TME investigation. With Foxn1nu .B6-Tg(CAG-EGFP) mice, our research demonstrated that host cell malignant transformation and tumor/host cell fusion play an important role in tumor progression.

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing.

BACKGROUND: Marek's disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. RESULTS: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. CONCLUSIONS: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Type 2 diabetes susceptibility genes on mouse chromosome 11 under high sucrose environment.

BACKGROUND: Both genetic and environmental factors contribute to type 2 diabetes development. We used consomic mice established from an animal type 2 diabetes model to identify susceptibility genes that contribute to type 2 diabetes development under specific environments. We previously established consomic strains (C3H-Chr 11NSY and C3H-Chr 14NSY) that possess diabetogenic Chr 11 or 14 of the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of spontaneous type 2 diabetes, in the genetic background of C3H mice. To search genes contribute to type 2 diabetes under specific environment, we first investigated whether sucrose administration deteriorates type 2 diabetes-related traits in the consomic strains. We dissected loci on Chr 11 by establishing congenic strains possessing different segments of NSY-derived Chr 11 under sucrose administration. RESULTS: In C3H-Chr 11NSY mice, sucrose administration for 10 weeks deteriorated hyperglycemia, insulin resistance, and impaired insulin secretion, which is comparable to NSY mice with sucrose. In C3H-Chr 14NSY mice, sucrose administration induced glucose intolerance, but not insulin resistance and impaired insulin secretion. To dissect the gene(s) existing on Chr 11 for sucrose-induced type 2 diabetes, we constructed four novel congenic strains (R1, R2, R3, and R4) with different segments of NSY-derived Chr 11 in C3H mice. R2 mice showed marked glucose intolerance and impaired insulin secretion comparable to C3H-Chr 11NSY mice. R3 and R4 mice also showed impaired insulin secretion. R4 mice showed significant decreases in white adipose tissue, which is in the opposite direction from parental C3H-Chr 11NSY and NSY mice. None of the four congenic strains showed insulin resistance. CONCLUSIONS: Genes on mouse Chr 11 could explain glucose intolerance, impaired insulin secretion, insulin resistance in NSY mice under sucrose administration. Congenic mapping with high sucrose environment localized susceptibility genes for type 2 diabetes associated with impaired insulin secretion in the middle segment (26.0-63.4 Mb) of Chr 11. Gene(s) that decrease white adipose tissue were mapped to the distal segment of Chr 11. The identification of diabetogenic gene on Chr 11 in the future study will facilitate precision medicine in type 2 diabetes by controlling specific environments in targeted subjects with susceptible genotypes.

[Effect of the C1473G Polymorphic Variant of the Tryptophan Hydroxylase 2 Gene and Photoperiod Length on the Dopamine System of the Mouse Brain].

A decrease in the light in autumn and winter causes depression like seasonal affective disorders (SAD) in sensitive patients, in which the serotonin (5-HT) and dopamine (DA) brain mediator systems are involved. We studied the interaction of the 5-HT and DA brain systems in an experimental SAD model in sexually mature male mice of the congenic B6-1473C and B6-1473G lines with high and low activity of tryptophan hydroxylase 2, a key enzyme of 5-HT synthesis in the brain. Mice of each line (divided into two groups of eight individuals) were kept for 30 days in standard (14 h light/10 h dark) and short (4 h light/20 h dark) daylight. The presence of the C1473G variant in the tryptophan hydroxylase 2 gene did not affect the expression of key genes of DA system: Drd1, Drd2, Scl6a3, Th, and Comt, that encode the D1 and D2 receptors, dopamine transporter, tyrosine hydroxylase, and catechol-o-methyltransferase, respectively. A decrease in the level of DA in the midbrain, as well as of its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, was detected in B6-1473G mice. Keeping mice in short daylight did not affect expression of the Drd1 gene in all brain structures nor the expression of the Slc6a3 and Th genes in the midbrain. Drd2 expression increased in the midbrain and decreased in the hippocampus, where Comt expression increased. An increase in DA level in the midbrain and DOPAC in the striatum was detected in mice kept in short daylight. This indicates the involvement of the brain's DA system in the reaction to a decrease in daylight duration. No statistically significant effect of the interaction between the presence of the C1473G variant and daylight length on indicators of the activity of DA system was detected. No reasons were found to assert that this polymorphism determines the observed reaction of the brain DA system in keeping of animals under short daylight conditions.

Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data.

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

Genetic characterization of early renal changes in a novel mouse model of diabetic kidney disease.

Genetic factors influence susceptibility to diabetic kidney disease. Here we mapped genes mediating renal hypertrophic changes in response to diabetes. A survey of 15 mouse strains identified variation in diabetic kidney hypertrophy. Strains with greater (FVB/N(FVB)) and lesser (C57BL/6 (B6)) responses were crossed and diabetic F2 progeny were characterized. Kidney weights of diabetic F2 mice were broadly distributed. Quantitative trait locus analyses revealed diabetic mice with kidney weights in the upper quartile shared alleles on chromosomes (chr) 6 and 12; these loci were designated as Diabetic kidney hypertrophy (Dkh)-1 and -2. To confirm these loci, reciprocal congenic mice were generated with defined FVB chromosome segments on the B6 strain background (B6.Dkh1/2f) or vice versa (FVB.Dkh1/2b). Diabetic mice of the B6.Dkh1/2f congenic strain developed diabetic kidney hypertrophy, while the reciprocal FVB.Dkh1/2b congenic strain was protected. The chr6 locus contained the candidate gene; Ark1b3, coding aldose reductase; the FVB allele has a missense mutation in this gene. Microarray analysis identified differentially expressed genes between diabetic B6 and FVB mice. Thus, since the two loci identified by quantitative trait locus mapping are syntenic with regions identified for human diabetic kidney disease, the congenic strains we describe provide a valuable new resource to study diabetic kidney disease and test agents that may prevent it.

Subcongenic analysis of a quantitative trait locus affecting body weight and glucose metabolism in zinc transporter 7 (znt7)-knockout mice.

BACKGROUND: A genome-wide mapping study using male F2 zinc transporter 7-knockout mice (znt7-KO) and their wild type littermates in a mixed 129P1/ReJ (129P1) and C57BL/6J (B6) background identified a quantitative trait locus (QTL) on chromosome 7, which had a synergistic effect on body weight gain and fat deposit with the znt7-null mutation. RESULTS: The genetic segment for body weight on mouse chromosome 7 was investigated by newly created subcongenic znt7-KO mouse strains carrying different lengths of genomic segments of chromosome 7 from the 129P1 donor strain in the B6 background. We mapped the sub-QTL for body weight in the proximal region of the previously mapped QTL, ranging from 47.4 to 64.4 megabases (Mb) on chromosome 7. The 129P1 donor allele conferred lower body weight gain and better glucose handling during intraperitoneal glucose challenge than the B6 allele control. We identified four candidate genes, including Htatip2, E030018B13Rik, Nipa1, and Atp10a, in this sub-QTL using quantitative RT-PCR and cSNP detection (single nucleotide polymorphisms in the protein coding region). CONCLUSIONS: This study dissected the genetic determinates of body weight and glucose metabolism in znt7-KO mice. The study demonstrated that a 17-Mb long 129P1 genomic region on mouse chromosome 7 conferred weight reduction and improved glucose tolerance in znt7-KO male mice. Among the four candidate genes identified, Htatip2 is the most likely candidate gene involved in the control of body weight based on its function in regulation of lipid metabolism. The candidate genes discovered in this study lay a foundation for future studies of their roles in development of metabolic diseases, such as obesity and type 2 diabetes.

Early production of offspring by in vitro fertilization using first-wave spermatozoa from prepubertal male mice.

Mature male mice (aged 10-12 weeks or older) are conventionally used for in vitro fertilization (IVF) in order to achieve high fertilization rates (e.g., > 70%). Here, we sought to determine the earliest age at which male mice (C57BL/6J strain) can be used efficiently for producing offspring via IVF. Because we noted that the addition of reduced glutathione (GSH) to the IVF medium significantly increased the fertilizing ability of spermatozoa from prepubertal males, we used this IVF protocol for all experiments. Spermatozoa first reached the caudal region of the epididymides at day 35; however, they were unable to fertilize oocytes. Caudal epididymal spermatozoa first became competent for oocyte fertilization at day 37, albeit at a low rate (2.9%). A high fertilization rate (72.0%) was obtained at day 40, and 52.4% of the embryos thus obtained developed into offspring after embryo transfer. Moreover, we found that corpus epididymal spermatozoa in prepubertal mice could fertilize oocytes; however, the fertilization rates were always < 50%, regardless of the age of the males. Caput epididymal spermatozoa failed to fertilize oocytes irrespective of the age of the males. Therefore, we propose that caudal epididymal spermatozoa from male mice aged 40 days can be efficiently used for IVF, to obtain offspring in the shortest attainable time. This protocol will reduce the turnover time required for the generation of mice by ~1 month compared with that of the conventional IVF protocol.

Conserved 33-kb haplotype in the MHC class III region regulates chronic arthritis.

Genome-wide association studies have revealed many genetic loci associated with complex autoimmune diseases. In rheumatoid arthritis (RA), the MHC gene HLA-DRB1 is the strongest candidate predicting disease development. It has been suggested that other immune-regulating genes in the MHC contribute to the disease risk, but this contribution has been difficult to show because of the strong linkage disequilibrium within the MHC. We isolated genomic regions in the form of congenic fragments in rats to test whether there are additional susceptibility loci in the MHC. By both congenic mapping in inbred strains and SNP typing in wild rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in the MHC-III region, which regulates the onset, severity, and chronicity of arthritis. The Ltab-Ncr3 haplotype consists of five polymorphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb (lymphotoxin-ß), Lst1 (leukocyte-specific transcript 1), and Ncr3 (natural cytotoxicity-triggering receptor 3). Significant correlation in the expression of the Ltab-Ncr3 genes suggests that interaction of these genes may be important in keeping these genes clustered together as a conserved haplotype. We studied the arthritis association and the spliceo-transcriptome of four different Ltab-Ncr3 haplotypes and showed that higher Ltb and Ncr3 expression, lower Lst1 expression, and the expression of a shorter splice variant of Lst1 correlate with reduced arthritis severity in rats. Interestingly, patients with mild RA also showed higher NCR3 expression and lower LST1 expression than patients with severe RA. These data demonstrate the importance of a conserved haplotype in the regulation of complex diseases such as arthritis.

Autoimmune-Disease-Prone NOD Mice Help To Reveal a New Genetic Locus for Reducing Pulmonary Disease Caused by Mycoplasma pulmonis.

Mycoplasmas are bacterial pathogens of a range of animals, including humans, and are a common cause of respiratory disease. However, the host genetic factors that affect resistance to infection or regulate the resulting pulmonary inflammation are not well defined. We and others have previously demonstrated that nonobese diabetic (NOD) mice can be used to investigate disease loci that affect bacterial infection and autoimmune diabetes. Here we show that NOD mice are more susceptible than C57BL/6 (B6) mice to infection with Mycoplasma pulmonis, a natural model of pulmonary mycoplasmosis. The lungs of infected NOD mice had higher loads of M. pulmonis and more severe inflammatory lesions. Moreover, congenic NOD mice that harbored different B6-derived chromosomal intervals enabled identification and localization of a new mycoplasmosis locus, termed Mpr2, on chromosome 13. These congenic NOD mice demonstrated that the B6 allele for Mpr2 reduced the severity of pulmonary inflammation caused by infection with M. pulmonis and that this was associated with altered cytokine and chemokine concentrations in the infected lungs. Mpr2 also colocalizes to the same genomic interval as Listr2 and Idd14, genetic loci linked to listeriosis resistance and autoimmune diabetes susceptibility, respectively, suggesting that allelic variation within these loci may affect the development of both infectious and autoimmune disease.

A congenic line of the C57BL/6J mouse strain that is proficient in melatonin synthesis.

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from nonfunctional alleles of the genes coding 2 key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed.

Characterization of Copy Number Variation's Potential Role in Marek's Disease.

Marek's Disease (MD) is a highly contagious pathogenic and oncogenic disease primarily affecting chickens. Chicken Lines 63 and 72, as well as their recombinant congenic strains (RCS) with varied susceptibility to MD, are ideal models to study the complex mechanisms of genetic resistance to MD. In this study, we investigated copy number variation (CNV) in these inbred chicken lines using the Affymetrix Axiom HD 600 K SNP genotyping array. We detected 393 CNV segments across all ten chicken lines, of which 12 CNVs were specifically identified in Line 72. We then assessed genetic structure based on CNV and observed markedly different patterns. Finally, we validated two deletion events in Line 72 and correlated them with genes expression using qPCR and RNA-seq, respectively. Our combined results indicated that these two CNV deletions were likely to contribute to MD susceptibility.

High-resolution mapping of a novel rat blood pressure locus on chromosome 9 to a region containing the Spp2 gene and colocalization of a QTL for bone mass.

Through linkage analysis of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR), a blood pressure (BP) quantitative trait locus (QTL) was previously located on rat chromosome 9. Subsequent substitution mapping studies of this QTL revealed multiple BP QTLs within the originally identified logarithm of odds plot by linkage analysis. The focus of this study was on a 14.39 Mb region, the distal portion of which remained unmapped in our previous studies. High-resolution substitution mapping for a BP QTL in the setting of a high-salt diet indicated that an SHR-derived congenic segment of 787.9 kb containing the gene secreted phosphoprotein-2 (Spp2) lowered BP and urinary protein excretion. A nonsynonymous G/T polymorphism in the Spp2 gene was detected between the S and S.SHR congenic rats. A survey of 45 strains showed that the T allele was rare, being detected only in some substrains of SHR and WKY. Protein modeling prediction through SWISSPROT indicated that the predicted protein product of this variant was significantly altered. Importantly, in addition to improved cardiovascular and renal function, high salt-fed congenic animals carrying the SHR T variant of Spp2 had significantly lower bone mass and altered bone microarchitecture. Total bone volume and volume of trabecular bone, cortical thickness, and degree of mineralization of cortical bone were all significantly reduced in congenic rats. Our study points to opposing effects of a congenic segment containing the prioritized candidate gene Spp2 on BP and bone mass.

Pappa2 is linked to salt-sensitive hypertension in Dahl S rats.

A 1.37 Mbp region of chromosome 13 previously identified by exclusion mapping was consistently associated with a reduction of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. This region contained five genes that were introgressed from the salt-insensitive Brown Norway (BN) rat. The goal of the present study was to further narrow that region to identify the gene(s) most likely to protect from salt-induced hypertension. The studies yielded a subcongenic SS rat strain containing a 0.71 Mbp insert from BN (26-P strain) in which salt-induced hypertension was reduced by 24 mmHg. The region contained two protein-coding genes (Astn1 and Pappa2) and a microRNA (miR-488). Pappa2 mRNA in the renal cortex of the protected 26-P was 6- to 10-fold greater than in SS fed a 0.4% NaCl diet but was reduced to levels observed in SS when fed 8.0% NaCl diet for 7 days. Compared with brain nuclei (NTS, RVLM, CVLM) and the adrenal gland, Pappa2 in the renal cortex was the only gene found to be differentially expressed between SS and 26-P and that responded to changes of salt diet. Immunohistochemistry studies found Pappa2 localized in the cytosol of the epithelial cells of the cortical thick ascending limbs. In more distal segments of the renal tubules, it was observed within tubular lumens and most notably bound to the apical membranes of the intercalated cells of collecting ducts. We conclude that we have identified a variant form of Pappa2 that can protect against salt-induced hypertension in the Dahl S rat.