Consomic Strains Research Articles

Phenotype and gene signature of testicular tumors in 129.MOLF-Chr19 mice resemble human teratomas.

Testicular germ cell tumor (TGCT) is the most common type of tumor in young men. Type II germ cell tumors including postpubertal-type teratomas are derived from the germ cell neoplasia in situ (GCNIS), whereas prepubertal-type teratomas arise independently of the GCNIS. The consomic mouse strain 129.MOLF-Chr19 (M19) is a suitable murine model of such tumors, but its characterization remains incomplete. Here, we interrogated the suitability of testicular tumors in M19 mice as a model of human TGCT by analyzing their histological features and gene expression signature. Testes collected from M19 mice of different ages were categorized by macroscopic appearance based on size and the degree of suspected tumorigenesis. Histological sections from selected tumors were stained with Hematoxylin and Eosin, and expression of genes associated with tumorigenesis was determined in frozen tissue samples from a large range of tumors of different subclasses using RT-qPCR and Fluidigm Dynamic Arrays. Macroscopically, testicular specimens appeared very heterogeneous concerning size and signs indicating the presence of a tumor. Histological analysis confirmed the development of teratomas with areas of cells corresponding to all three germ cell layers. Gene expression analyses indicated upregulation of markers related to proliferation, vascular invasive potential and pluripotency, and revealed a strong correlation of gene expression with tumor size and a significant intercorrelation of individual genes. TGCT in M19 mice is reminiscent of human testicular teratomas presenting with areas of cells derived from all germ layers and showing a typical gene signature. We thus confirm that these mice can serve as a suitable murine model of pure teratomas for preclinical research.

Recessive mutation on mouse chromosome 13 associated with abnormal hair texture and cardiomyopathy.

An autosomal recessive mutation (aht) associated with abnormal hair texture and cardiomyopathy spontaneously arose in the Y-chromosome consomic mouse strain DH-Chr YSS . The aht/aht mouse phenotypes closely resembled those of rul/rul mice, which were caused by a mutation in desmoplakin (Dsp) on chromosome 13. Quantitative trait locus (QTL) mapping using (DDD/Sgn × DH-Chr YSS -aht heterozygotes) F2 mice demonstrated that aht is contiguous with Dsp on chromosome 13. However, no nucleotide changes were identified in the coding region of Dsp in aht/aht mice by whole-exome sequencing. Therefore, the molecular nature of the aht mutation remains unclear. Nevertheless, aht/aht mice may serve as a new model for human diseases that are accompanied by abnormalities in the integumental and cardiovascular systems, including Carvajal-Huerta syndrome.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13Gy partial body irradiation at 42- and 90days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Descending Control of Nociception Poorly Predicts the Development of Persistent Postsurgical Pain-like Behavior in Consomic Dahl S Rat Strains.

Chronic postsurgical pain is a poorly recognized outcome of surgery where patients experience pain long after healing from the surgical insult. Descending control of nociception, a phenomenon whereby application of a strong nociceptive stimulus to one part of the body of animals inhibits pain in remote body regions, offers one strategy to identify a propensity to develop chronic postsurgical pain-like behavior. Here, consomic rat panel was used to test the hypothesis that pain persistence is mechanistically linked to ineffective descending control of nociception. Male and female Brown Norway, Dahl S, and eight consomic strains (SS-xBN) were used to determine the presence of chronic postsurgical pain-like behaviors by using paw-withdrawal threshold evaluation (von Frey method) in the area adjacent to a hind paw plantar incision. Descending control of nociception was assessed by measuring hind paw-withdrawal thresholds (Randall-Selitto method) after capsaicin (125 µg) injection into a forepaw. Consomic rats were developed by introgressing individual Brown Norway chromosomes on the Dahl S rat genetic background, as Dahl S rats lack preoperative descending control of nociception. Substitution of several chromosomes from the "pain-resistant" Brown Norway to the "pain-prone" Dahl S/Medical College of Wisconsin reduced mechanical nociceptive sensitivity and increased endogenous pain modulation capacity by differing degrees. Statistical modeling of these data revealed that descending control of nociception is a poor general predictor of the propensity to develop chronic postsurgical pain-like behavior (poor fit for model 1). However, a significant strain-by-descending control of nociception interaction was revealed (model 3, -2*log likelihood; 550.668, -2ll change; 18.093, P = 0.034) with SS-13BN and SS-15BN strains showing a negative descending control of nociception relationship with chronic postsurgical pain-like behavior. Descending control of nociception poorly predicted which rat strains developed chronic postsurgical pain-like behavior despite controlling for genetic, environmental, and sex differences. Two consomic strains that mimic clinical chronic postsurgical pain criteria and display a strong negative correlation with descending control of nociception were identified, offering novel candidates for future experiments exploring mechanisms that lead to chronic postsurgical pain.

Neonatal lethality of mouse A/J-7SM consomic strain is caused by an insertion mutation in the Dchs1 gene.

Homosomic mice of the A/J-7SM consomic mouse strain that introduced the entire chromosome 7 (Chr 7) of SM/J into the A/J strain exhibited neonatal lethality. We tentatively maintained segregating inbred strains (A/J-7ASM and A/J-7DSM) in which the central portion of Chr 7 was heterozygous for the A/J and SM/J strains, and the centromeric and telomeric sides of Chr 7 were homozygous for the SM/J strain, instead of the A/J-7SM strain. Based on the chromosomal constitution of Chr 7 in A/J-7ASM and A/J-7DSM mice, the causative gene for neonatal lethality in homosomic mice was suggested to be located within an approximately 1.620Mb region between D7Mit125 (104.879Mb) and D7Mit355 (106.499Mb) on Chr 7. RT-PCR analysis revealed that homosomic mice lacked dachsous cadherin-related 1 (Dchs1), which is located within the D7Mit125 to D7Mit355 region and functions in the regulation of planar cell polarity. Screening for mutations in Dchs1 indicated that homosomic mice possessed an early transposable (ETn)-like sequence in intron 1 of Dchs1. Moreover, an allelism test between Dchs1 ETn-like-insertion alleles detected in homosomic mice and CRISPR/Cas9-induced Dchs1 deletion alleles revealed that Dchs1 is a causative gene for neonatal lethality in homosomic mice. Based on these results, we concluded that in the A/J-7SM strain, ETn-like elements were inserted into intron 1 of SM/J-derived Dchs1 during strain development, which dramatically reduced Dchs1 expression, thus resulting in neonatal lethality in homosomic mice. Additionally, it was suggested that the timing of lethality in Dchs1 mutant mice is influenced by the genetic background.

Sex differences in susceptibility to influenza A virus infection depend on host genotype.

Infection with the respiratory pathogen influenza A virus (IAV) causes significant morbidity and mortality each year. While host genotype is thought to contribute to severity of disease, naturally occurring genetic determinants remain mostly unknown. Moreover, more severe disease is seen in women compared with men, but genetic mechanisms underlying this sex difference remain obscure. Here, using IAV infection in a mouse model of naturally selected genetic diversity, namely C57BL6/J (B6) mice carrying chromosomes (Chr) derived from the wild-derived and genetically divergent PWD/PhJ (PWD) mouse strain (B6.ChrPWD consomic mice), we examined the effects of genotype and sex on severity of IAV-induced disease. Compared with B6, parental PWD mice were completely protected from IAV-induced disease, a phenotype that was fully recapitulated in the B6.Chr16PWD strain carrying the PWD-derived allele of Mx1. In contrast, several other consomic strains, including B6.Chr3PWD and B6.Chr5PWD, demonstrated greatly increased susceptibility. Notably, B6.Chr5PWD and B6.ChrX.3PWD strains, the latter carrying the distal one-third of ChrX from PWD, exhibited increased morbidity and mortality specifically in male but not female mice. Follow up analyses focused on B6 and B6.ChrX.3PWD strains demonstrated moderately elevated viral load in B6.ChrX3PWD male, but not female mice. Transcriptional profiling demonstrated genotype- and sex-specific gene expression profiles in the infected lung, with male B6.ChrX.3 mice exhibiting the most significant changes, including upregulation of a proinflammatory gene expression program associated with myeloid cells, and altered sex-biased expression of several X-linked genes that represent positional candidates, including Tlr13 and Slc25a53. Taken together, our results identify novel loci on autosomes and the X chromosome regulating IAV susceptibility and demonstrate that sex differences in IAV susceptibility are genotype-dependent, suggesting that future genetic association studies need to consider sex as a covariate.

Identification of a novel mutant allele of LIM homeobox transcription factor 1 alpha (dreher) in mice.

A male mouse exhibiting bidirectional circling behavior was identified in a Y-chromosome consomic strain known as DH-Chr YRR . The putative mutation responsible for the circling behavior was inherited in an autosomal recessive manner and was termed circ. To identify its causative gene, we performed exome sequencing; of the 34 candidates discovered, we found a novel nonsynonymous single nucleotide variation in LIM homeobox transcription factor 1 alpha (Lmx1a) (c.394G > C, p.Gly132Arg). The genetic linkage between Lmx1a and circ was confirmed in (♀BALB/cA × ♂DH-Chr YRR -circ/circ) F2 and (♀C57BL/6J × ♂DH-Chr YRR -circ/circ) F2 mice. The Lmx1a mutation led to many abnormalities that affected growth, pigmentation, reproduction, and cerebellar morphology. We showed that (♀BALB/cA × ♂DH-Chr YRR -circ/circ) F2 -circ/circ mice demonstrated significantly lower body mass than the F2 -+/? mice. Unlike the F2 -+/? mice, few (♀C57BL/6J × ♂DH-Chr YRR -circ/circ) F2 -circ/circ mice exhibited a belly spot. The circ/circ females were also invariably sterile, probably because of an underdeveloped uterus. Moreover, the circ/circ mice presented fewer cerebellar granule cells with lower density than the F2 -+/? mice. Although non-complementation between circ and the known Lmx1a mutant alleles remains unconfirmed, the coisogenic nature of circ strongly suggests that it is a novel variant of Lmx1a, previously known as dreher. Therefore, we have assigned the gene symbol Lmx1adr-circ to circ. In addition to Lmx1adr-J and Lmx1adr-kjmi , Lmx1adr-circ is the third allele that causes a missense mutation within LIM domains. Identification of missense mutations is necessary to specify the critical residues for abrogating the in vivo functions of LMX1A.

Integrative genomic analysis of blood pressure and related phenotypes in rats.

ABSTRACTDespite remarkable progress made in human genome-wide association studies, there remains a substantial gap between statistical evidence for genetic associations and functional comprehension of the underlying mechanisms governing these associations. As a means of bridging this gap, we performed genomic analysis of blood pressure (BP) and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP), both of which are unique genetic models of severe hypertension and cardiovascular complications. By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n=1415), fine congenic mapping (maximum n=8704), pharmacological intervention and comparative analysis with transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY); e.g. inter-strain BP differences between SHRSP and WKY could be largely explained by an aggregate of BP changes in seven SHRSP-derived consomic strains. We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this study, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein–kinin/renin–angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. In combination with a pharmacological intervention, we provide in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, in a rat polygenic hypertension model.

Genetic controls of Tas1r3-independent sucrose consumption in mice.

We have previously used crosses between C57BL/6ByJ (B6) and 129P3/J (129) inbred strains to map a quantitative trait locus (QTL) on mouse chromosome (Chr) 4 that affects behavioral and neural responses to sucrose. We have named it the sucrose consumption QTL 2 (Scon2), and shown that it corresponds to the Tas1r3 gene, which encodes a sweet taste receptor subunit TAS1R3. To discover other sucrose consumption QTLs, we have intercrossed B6 inbred and 129.B6-Tas1r3 congenic mice to produce F2 hybrids, in which Scon2 (Tas1r3) does not segregate, and hence does not contribute to phenotypical variation. Chromosome mapping using this F2 intercross identified two main-effect QTLs, Scon3 (Chr9) and Scon10 (Chr14), and an epistatically interacting QTL pair Scon3 (Chr9)-Scon4 (Chr1). Using serial backcrosses, congenic and consomic strains, we conducted high-resolution mapping of Scon3 and Scon4 and analyzed their epistatic interactions. We used mice with different Scon3 or Scon4 genotypes to understand whether these two QTLs influence sucrose intake via gustatory or postoral mechanisms. These studies found no evidence for involvement of the taste mechanisms, but suggested involvement of energy metabolism. Mice with the B6 Scon4 genotype drank less sucrose in two-bottle tests, and also had ahigher respiratory exchange ratio and lower energy expenditure under basal conditions (when they had only chow and water available). Our results provide evidence that Scon3 and Scon4 influence mouse-to-mouse variation in sucrose intake and that both likely act through a common postoral mechanism.

Abstract IA004: Genetic variants in the tumor microenvironment alter radiation responses in breast cancer

Abstract Objectives: The tumor microenvironment (TME) can impact breast cancer tumor growth, progression, and treatment responses. Data suggests that genetic variants in not only breast cancer cells, but also in the TME, can also alter these processes. We have utilized a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel to identify genetic variants in the TME that affect radiation sensitivity. Materials/Methods: Human triple negative breast cancer MDA-MD-231 cells were implanted into immunodeficient (IL2RG KO) consomic rat strains that are genetically identical except for chromosome 3 is inherited from a separate strain (SS and SS.BN3 strains). On day 10, tumors were treated with 3 daily ionizing radiation (IR) treatments of 4 Gy or sham, and tumor growth was monitored. Tumors were also harvested for hypoxia staining using pimonidazole or for RNA-seq. RNA-Seq was performed and a custom SSRS protocol was used to align both rat and human transcripts. This yielded transcript and gene level estimated fold-change and adjusted p-values for human- and rat-derived transcripts separately. E077 mammary tumor cells were implanted into adult female immune competent C57/Bl6 mice. On day 5, tumors were treated with 5 daily IR treatments of 5 Gy or sham. Either vehicle or a mAb to the Notch ligand Dll4 (Genentech) was given twice weekly. Chi-square, Fisher’s exact, and Kolmogorov-Smirnov tests and empirical cumulative distribution plots for differential expression significance values were performed. Results: Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor IR sensitivity, as human breast cancer xenografts in the consomic strain (SS.BN3) were significantly more IR sensitive than SS rat strain tumors (supra-additive). Vascular gene pathways were differentially expressed, and tumor vascular phenotypes were distinct, with SS.BN3 tumors with increased but poorly functioning blood vessels. Hypoxia was similar at baseline, but increased in SS.BN3 tumors following IR. These results were consistent with less Dll4 expression in the SS.BN3 TME. The use of a Dll4-targeted mAb in mice demonstrated that targeting Dll4 enhanced mammary tumor IR responses. Conclusion: CXM demonstrated TME genetic variants can affect IR sensitivity of genetically identical tumor cells. Using SSRS, we identified candidate genes on rat chromosome 3 that may potentially influence IR sensitivity, and our studies ultimately led to identification of the Notch ligand Dll4 as a target to enhance breast cancer IR responses. Future studies will investigate the possibility of the Dll4 pathway as a therapeutic target, as well as interrogate other pathways responsible for changes in IR sensitivity seen in the CXM model. Determining TME factors that affect the IR sensitivity will allow more tailored and effective treatments. Citation Format: Carmen Bergom, Michael W. Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Rachel A. Schlaak, Shirng-Wern Tsaih, Michael J. Flister. Genetic variants in the tumor microenvironment alter radiation responses in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA004.

PWD/Ph-Encoded Genetic Variants Modulate the Cellular Wnt/β-Catenin Response to Suppress Apc Min-Triggered Intestinal Tumor Formation.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone Apc Min/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APC Min offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin-driven and stem cell-specific gene expression in the presence of Apc Min or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development. SIGNIFICANCE: These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

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

BackgroundBoth 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.ResultsIn 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.ConclusionsGenes 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.

Sperm quality, aggressiveness and generation turnover may facilitate unidirectional Y chromosome introgression across the European house mouse hybrid zone.

The widespread and locally massive introgression of Y chromosomes of the eastern house mouse (Mus musculus musculus) into the range of the western subspecies (M. m. domesticus) in Central Europe calls for an explanation of its underlying mechanisms. Given the paternal inheritance pattern, obvious candidates for traits mediating the introgression are characters associated with sperm quantity and quality. We can also expect traits such as size, aggression or the length of generation cycles to facilitate the spread. We have created two consomic strains carrying the non-recombining region of the Y chromosome of the opposite subspecies, allowing us to study introgression in both directions, something impossible in nature due to the unidirectionality of introgression. We analyzed several traits potentially related to male fitness. Transmission of the domesticus Y onto the musculus background had negative effects on all studied traits. Likewise, domesticus males possessing the musculus Y had, on average, smaller body and testes and lower sperm count than the parental strain. However, the same consomic males tended to produce less- dissociated sperm heads, to win more dyadic encounters, and to have shorter generation cycles than pure domesticus males. These data suggest that the domesticus Y is disadvantageous on the musculus background, while introgression in the opposite direction can confer a recognizable, though not always significant, selective advantage. Our results are thus congruent with the unidirectional musculus → domesticus Y chromosome introgression in Central Europe. In addition to some previous studies, they show this to be a multifaceted phenomenon demanding a multidisciplinary approach.

Genetic regulation of nitric oxide-dependent mitochondrial metabolism and immune function in dendritic cells

Abstract Dendritic cell (DC) activation is characterized by a rapid “metabolic switch” towards increased glycolysis that supports maturation. Sustained glycolysis post-activation is a requirement for survival in DC subsets that express inducible nitric oxide synthase (iNOS), due to the production of nitric oxide (NO) which acts as an inhibitor of mitochondrial respiration. Long-term metabolic reprogramming in activated DCs has almost exclusively been studied in the strain C57BL/6, which experience loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we generated and compared cells from C57BL/6 mice to the wild-derived genetically diverse PWD/PhJ (PWD) strain. We show attenuated iNOS induction and significantly lower NO production in PWD DCs. Decreased NO production allows PWD DCs to maintain mitochondrial respiration following activation, and significantly alters their glucose-dependent carbon flux through the TCA cycle compared to C57BL/6 DCs. We utilize a consomic strain which includes the PWD chromosome segment containing the iNOS locus on an otherwise C57BL/6 genetic background to demonstrate that intrinsic differences in the iNOS locus drive differential iNOS regulation. We demonstrate that differential iNOS regulation amongst these strains impacts DC post-activation survival and immune effector functions. These studies provide a more comprehensive understanding of NO-driven metabolic reprogramming in the murine myeloid lineage and establish a natural genetic model for investigating the contribution of NO in regulating the interplay between DC metabolism and immune function.

Heritable modifiers of the tumor microenvironment influence nanoparticle uptake, distribution and response to photothermal therapy.

We report the impact of notch-DLL4-based hereditary vascular heterogeneities on the enhanced permeation and retention (EPR) effect and plasmonic photothermal therapy response in tumors.Methods: We generated two consomic rat strains with differing DLL4 expression on 3rd chromosome. These strains were based on immunocompromised Salt-sensitive or SSIL2Rγ- (DLL4-high) and SS.BN3IL2Rγ- (DLL4-low) rats with 3rd chromosome substituted from Brown Norway rat. We further constructed three novel SS.BN3IL2Rγ- congenic strains by introgressing varying segments of BN chromosome 3 into the parental SSIL2Rγ- strain to localize the role of SSIL2Rγ- DLL4 on tumor EPR effect with precision. We synthesized multimodal theranostic nanoparticles (TNPs) based on Au-nanorods which provide magnetic resonance imaging (MRI), X-ray, and optical contrasts to assess image guided PTT response and quantify host specific therapy response differences in tumors orthotopically xenografted in DLL4-high and -low strains. We tested recovery of therapy sensitivity of PTT resistant strains by employing anti-DLL4 conjugated TNPs in two triple negative breast cancer tumor xenografts.Results: Host strains with high DLL4 allele demonstrated slightly increased tumor nanoparticle uptake but consistently developed photothermal therapy resistance compared to tumors in host strains with low DLL4 allele. Tumor micro-environment with low DLL4 expression altered the geographic distribution of nanoparticles towards closer proximity with vasculature which improved efficacy of PTT in spite of lower overall TNP uptake. Targeting TNPs to tumor endothelium via anti-DLL4 antibody conjugation improved therapy sensitivity in high DLL4 allele hosts for two triple negative human breast cancer xenografts.Conclusions: Inherited DLL4 expression modulates EPR effects in tumors, and molecular targeting of endothelial DLL4 via nanoparticles is an effective personalized nanomedicine strategy.

Y Chromosome, Hypertension and Cardiovascular Disease: Is Inflammation the Answer?

It is now becomingly increasingly evident that the functions of the mammalian Y chromosome are not circumscribed to the induction of male sex. While animal studies have shown variations in the Y are strongly accountable for blood pressure (BP), this is yet to be confirmed in humans. We have recently shown modulation of adaptive immunity to be a significant mechanism underpinning Y-chromosome-dependent differences in BP in consomic strains. This is paralleled by studies in man showing Y chromosome haplogroup is a significant predictor for coronary artery disease through influencing pathways of immunity. Furthermore, recent studies in mice and humans have shown that Y chromosome lineage determines susceptibility to autoimmune disease. Here we review the evidence in animals and humans that Y chromosome lineage influences hypertension and cardiovascular disease risk, with a novel focus on pathways of immunity as a significant pathway involved.

Interactions between the host genome and the gut microbiome determine susceptibility to CNS autoimmune disease

Abstract Multiple sclerosis (MS) is a chronic autoimmune central nervous system (CNS) disease and the leading cause of non-traumatic neurological disability in young adults. The etiology of MS is multifactorial and polygenic, with 30% of risk genetic and 70% environmental. Recent studies identified an imbalance in the human gut microbiome within MS patients, including depletion of the Lactobacillus genus. Animal models support a causal role of the gut microbiome in MS, though the mechanisms remain unclear. Here, we leverage natural genetic variation in wild-derived PWD/PhJ (PWD) mice as compared to classic C57BL/6J (B6) mice, modeling human genetic diversity to identify host genome and gut microbiome interactions governing disease susceptibility. In a model of MS, experimental autoimmune encephalomyelitis (EAE), 27 consomic strains carrying PWD chromosomes on the B6 background demonstrated that PWD-derived loci exhibit bidirectional and sex-specific effects on disease severity. 16S rRNA gene sequencing of the gut microbiome in consomic and parental strains identified strain-specific profiles and metabolic functions associated with differential susceptibility to EAE. Functionally, manipulation of the gut microbiome by co-housing or transplantation, demonstrated direct host genotype-dependent effects of the microbiome on EAE susceptibility, with transfer of signature Lactobacillus species associated with changes in circulating indole metabolites and altered EAE severity. These studies reveal the presence of complex bidirectional interactions between the host genome and gut microbiome that modulate susceptibility to CNS autoimmunity, and identify specific microbiome constituents as potential therapeutic candidates.

Identification of novel loci controlling IBD susceptibility utilizing the genetic diversity of wild-derived mice

Abstract Inflammatory bowel disease (IBD) is a complex disease that imposes a significant and growing health burden in industrialized nations. Recent genetic studies have identified multiple genetic associations with IBD, but the mechanisms underlying these associations are poorly understood. Animal models are needed to bridge this gap in knowledge, but conventional laboratory mouse strains lack the genetic diversity of human populations. To more accurately model human genetic diversity and to identify new genetic loci that control susceptibility to IBD, we utilized a panel of chromosome (Chr) substitution (consomic) strains, carrying chromosomes from the wild-derived and genetically divergent PWD/PhJ (PWD) strain on the commonly used C57BL/6J (B6) background. Two models of IBD were used, TNBS- and DSS-induced colitis. Multiple disease severity parameters were evaluated, including weight loss, disease activity, fecal Lipocalin-2 production, and histopathology. Strikingly, compared with B6 mice, wild-derived PWD mice were highly susceptible to TNBS-induced colitis, but resistant to DSS-induced colitis. Using consomic mice, we found several PWD-derived loci that exhibited significant effects on IBD susceptibility. Most pronounced was a locus on Chr2, which resulted in very high susceptibility to both DSS and TNBS colitis, while several other PWD-derived loci were protective. Altogether, our experiments identified novel IBD susceptibility loci, harboring genes that control susceptibility to various aspects of IBD pathogenesis, and underscoring the utility of wild-derived mouse genetics. Future studies will identify these genes and their functions, to generate new insights into IBD etiology and pathophysiology.

Verification That Mouse Chromosome 14 Is Responsible for Susceptibility to Streptozotocin in NSY Mice.

Introduction Streptozotocin- (STZ-) induced diabetes is under polygenic control, and the genetic loci for STZ susceptibility are mapped to chromosome (Chr) 11 in Nagoya-Shibata-Yasuda (NSY) mice. In addition to Chr11, other genes on different chromosomes may contribute to STZ susceptibility in NSY mice. The aim of this study was to determine whether NSY-Chr14 contributes to STZ susceptibility and contains the STZ-susceptible region. Materials and Methods A consomic C3H-14NSY strain (R0: homozygous for NSY-derived whole Chr14 on the control C3H background), two congenic strains (R1: the region retained proximal and middle segments of NSY-Chr14 and R2: the region retained a proximal segment of NSY-Chr14), and parental NSY and C3H mice were intraperitoneally injected with a single injection of STZ at a dose of 175 mg/kg body weight at 12 weeks of age. Blood glucose levels and body weights were measured at days 0, 1, 2, 4, 5, 7, 8, and 14 after STZ injection. At day 14 after STZ injection, pancreata were dissected and fixed. Results After STZ injection, blood glucose levels were significantly higher in R0 mice than in C3H mice. However, blood glucose levels in R0 mice were not as severely affected as those in NSY mice. In R1 and R2 mice, blood glucose levels were similar to those in C3H mice and were significantly lower than those in R0 mice. Body weights were decreased in NSY and R0 mice; however, this change was not observed in R1, R2, and C3H mice. Although islet tissues in all strains exhibited degeneration and cellular infiltration, histological changes in NSY and R0 mice were more severe than those in R1, R2, and C3H mice. Conclusions These data demonstrated that NSY-Chr14 was a STZ-susceptible chromosome and that STZ susceptibility was mapped to the distal segment of NSY-Chr14.

Abstract P292: Gene Editing Rat Resource Center: Rat Models for Heart, Lung, Blood, and Sleep Disorder Studies

Transgenesis and gene editing in the rat has produced gene-modified strains which can be used to validate a gene underlying a quantitative trait locus or human association, or to design follow up studies to define function and physiological roles of genes or sequence variants. We are currently in the fifth year of developing rat models for investigators to validate human variation and study function and mechanism for genes implicated in heart, lung and blood disorders. The Gene Editing Rat Resource Center (GERRC, http://rgd.mcw.edu/wg/custom_rats/gerrc ) is an NHLBI-funded R24 resource program to generate, distribute, and cryopreserve novel rat models to the research community. Investigator-initiated applications to produce novel genetically modified rat strains were received from laboratories across the world and reviewed by an external advisory board for scientific merit and potential broad interest to the heart, lung, blood, and sleep disorder research community. Custom knockout, knockin, and transgenic rat models continue to be developed. To date, we have transferred over 19,570 microinjected embryos, resulting in 4000+ live-born pups of which 3400+ have been screened for transgenesis or mutagenesis of the target gene. Of these, we have successfully generated ~630 pups containing transgenes or targeted mutations in 95 genes, distributed among 16 inbred, outbred, consomic and congenic rat strains frequently used in cardiovascular research. After confirming germline transmission, heterozygous breeders are distributed to the requesting investigator and then each model is made available to the rat research community. Sperm is cryopreserved to maintain a permanent source of these models. Collectively, the GERRC resource represents the largest collection of genetically modified rat models which are distributable to any investigator through a standard materials transfer agreement at the cost of rearing and shipping. We will discuss the general progress of genetic engineering in rats, current challenges for the field, and opportunities for future developments.