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Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate–activated protein kinase alpha signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis. Conclusion: NgBR is a specific negative regulator for LXRα-dependent hepatic lipogenesis. Loss of NgBR may be a potential trigger for inducing hepatic steatosis. (Hepatology 2016;64:1559-1576)

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.

Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to type IV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its in vivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagens I and IV was measured. VWF-dependent platelet adhesion to collagen IV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagen IV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagen IV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagen IV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen IV binding.

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd(-/-)) mice to term. Half of the Thbd(-/-) offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd(-/-) animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd(-/-) mice. However, Thbd(-/-) females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy

Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation via Akt pathway in zebrafish. Here, we further demonstrated the roles of NgBR in regulating vasculature development in mouse embryo and primitive blood vessel formation in embryoid body culture systems, respectively. Our results showed that NgBR homozygous knockout mice are embryonically lethal at E7.5 or earlier, and Tie2Cre-mediated endothelial cell-specific NgBR knockout (NgBR ecKO) mice die at E11.5 and have severe blood vessel assembly defects in embryo. In addition, mutant embryos exhibit dilation of cerebral blood vessel, resulting in thin-walled endothelial caverns. The similar vascular defects also were detected in Cdh5(PAC)-CreERT2 NgBR inducible ecKO mice. Murine NgBR gene-targeting embryonic stem cells (ESC) were generated by homologous recombination approaches. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but reduces the formation and branching of primitive blood vessels in embryoid body culture systems. Mechanistically, NgBR knockdown not only decreases both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases the expression of CCM1 and CCM2 proteins. Furthermore, we performed immunofluorescence (IF) staining of NgBR in human cerebral cavernous malformation patient tissue sections. The quantitative analysis results showed that NgBR expression levels in CD31 positive endothelial cells is significantly decreased in patient tissue sections. These results suggest that NgBR may be one of important genes coordinating the cerebral vasculature development.

The lack of rat embryonic stem cells (ESCs) and approaches for manipulation of their genomes have restricted the ability to create new genetic models and to explore the function of a single gene in complex diseases in the laboratory rat. The recent breakthrough in isolating germline-competent ESCs from rat and subsequent demonstration of gene knockout has propelled the field forward, but such tools do not yet exist for many disease-model rat strains. Here we derive new ESCs from several commonly used rat models including the Dahl Salt Sensitive (SS), the sequenced Brown Norway (BN), and Fischer (F344) rat and establish the first germline-competent ESCs from a hypertension disease model strain, the Fawn Hooded Hypertensive (FHH) rat. Genetic manipulations including transgenesis mediated by lentivirus, routine homologous recombination, and homologous recombination mediated by zinc-finger nucleases (ZFNs) were performed effectively in FHH rat ESCs. Our results showed these rat ESC lines, isolated from inner cell masses using mechanical splitting, had germline competency; the Pparg gene locus and homologous genomic region to the mouse Rosa26 locus can be targeted effectively in these rat ESCs. Furthermore, our results also demonstrated that ZFNs increased the efficiency of proper homologous recombination in FHH rat ESCs using targeting vectors with short homology arms. These rat ESC lines and advancements in genetic manipulation pave the way to novel genetic approaches in this valuable biomedical model species and for exploration of complex disease in these strains.

VWF binding to type 4 collagen has recently been shown to occur via the VWF A1 domain, but the clinical implications of this interaction remain to be elucidated. In humans, the R1399H sequence variant (2% of the Caucasian population is heterozygous for this variant) abrogates VWF binding to collagen 4, while select other VWF A1 domain variants decrease VWF binding to collagen 4 to varying degrees. We generated a murine model of 1399H VWF to investigate the in vitro and in vivo effects of this variant more thoroughly. Mice expressing the murine R1399H mutation were generated via gene targeting in ES cells, generation of germline-competent ES cell chimeras, and backcrossing the resultant mutant mice onto a C57Bl6 background. VWF antigen (VWF:Ag) and VWF collagen binding with murine type 4 collagen (VWF:CB4) were measured by ELISA (n≥5 for each group) on wild-type p.R1399 mice (WT), heterozygous p.R1399H mice (R1399H) and homozygous p.H1399 mice (1399HH) and compared to mice lacking VWF entirely (VWF -/-). Tail bleeding time was performed by clipping a 3 mm segment of tail, immersing in warm saline, and monitoring bleeding for a maximum of 10 minutes (n≥4 for each group). Blood loss was also measured during the tail bleeding time assay for each mouse. VWF-dependent platelet adhesion to collagen 4 was measured under flow using the Venaflux system. Channels were coated with murine type 4 collagen overnight. Blood was drawn from the inferior vena cava, anticoagulated with PPACK and heparin, and platelets labeled with mepacrine. Whole blood samples were run over the collagen 4-coated channel at a shear rate of 1111/sec (n≥3 for each group). Images at 180 seconds following the start of flow over the chip were analyzed using ImageJ. Breeding of heterozygous and homozygous 1399H mice was observed to follow normal Mendelian ratios, indicating no deleterious effects of the 1399H variant on viability. No spontaneous bleeding was observed for any of the offspring. VWF expression as measured by VWF:Ag was similar for WT, R1399H, and 1399HH mice, but VWF:CB4/VWF:Ag ratios were decreased for R1399H mice, to an average of 55% of WT and 1399HH mice, to an average of 15% of WT. Blood loss during tail bleeding was increased compared to WT for both R1399H and 1399HH mice by approximately 1.8 fold and 2.8 fold respectively. Bleeding time also increased, with an average bleeding time of 92 seconds in the WT mice, 177 seconds in the R1399H mice, and 235 seconds in the 1399HH mice. Previous studies have demonstrated that platelet adhesion to collagen 4 under flow is dependent on VWF, with VWF -/- mice demonstrating absence of platelet adhesion. Platelet adhesion to collagen 4 was diminished in both heterozygous and homozygous 1399H mice, with maximum platelet binding intensity 61% of WT for the R1399H mice and 28% of WT for the 1388HH mice. These figures closely approximate the static collagen 4 binding as seen by comparable VWF:CB4/VWF:Ag ratios and platelet adhesion under flow for the R1399H heterozygous and 1399HH homozygous mice. These results show that a decrease in VWF's ability to bind collagen 4 under static conditions corresponds to a decrease in binding under flow conditions, at least at the measured shear rate, and that this defect also translates to increased bleeding time and blood loss in a tail clip model. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen 4 binding. Since 2% of the US population is heterozygous for the p.R1399H sequence variation, defective VWF interaction with collagen 4 may be a relatively common cause of clinical bleeding and can be modeled in engineered mice. Disclosures Flood: CSL Behring: Consultancy; Baxter: Consultancy. Montgomery:Baxter: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Octapharma: Consultancy; Grifols: Consultancy; Biogen: Consultancy.

Human platelet alloantigens (HPAs) are caused by single nucleotide polymorphisms within functionally important platelet membrane glycoproteins. Antibodies against HPAs have been implicated in several clinically important alloimmune platelet disorders, including neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura, and platelet transfusion refractoriness. A single C29523T nucleotide substitution, resulting in a Leu33Pro amino acid polymorphism within PSI domain of the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes, and is the most commonly implicated HPA in alloimmune platelet disorders in Caucasian populations. Allogeneic mismatch between an HPA-1b homozygous mother and an HPA-1a-positive fetus can lead to maternal alloimmunization, with the production of anti-HPA-1a antibodies that cross the placenta, where they bind fetal platelets and endothelial cells. The resulting thrombocytopenia and damage to fetal endothelial cells results in NAIT. Existing animal models of NAIT involve either (1) injecting human platelets into NOD/SCID immunodeficient mice followed by intraperitoneal injections of human maternal alloantisera, or (2) transfusing wild-type mouse platelets into GPIIb-IIIa-deficient mice to generate an isoimmune, rather than an alloimmune, response. Neither of these models truly mimic NAIT, as only the kinetics of platelet clearance can be measured in the former model, while the latter is a model of iso-, rather than allo-, immunity. Mouse β3 contains neither Leu nor proline at residue 33 position, and human maternal anti-HPA-1a antibodies do not recognize the murine protein. Previous studies have shown, however, that substitution of only four amino acids within the PSI domain is sufficient for reconstituting the human anti-HPA-1a epitope into the murine protein. This laid the foundation for rational genomic editing to recreate the HPA-1a epitope in mice. Guide RNAs that target the ITGB3 gene near the region encoding amino acid 33 were cloned into the CRISPR expression plasmid, px459. A 200 bp single-stranded DNA oligonucleotide corresponding to murine β3, but containing nine nucleotide substitutions that introduced four amino acid substitutions (T30A, S32P, Q33L, and N39D) into the mouse β3 cDNA was used as a template for homology-directed repair (HDR) of the endogenous ITGB3 gene. These four amino acid substitutions (APLD) have previously been shown to be necessary for reconstituting the human anti-HPA-1a epitope in murine β3. The CRISPR plasmid and HDR repair template were co-injected into fertilized mouse zygotes, the progeny of which were then screened by diagnostic restriction enzyme digestion for successful homozygous integration of the HDR repair template. That platelets from the APLD mice expressed the human HPA-1 alloantigenic epitope was confirmed by flow-cytometry and Western-blot analysis using the HPA-1a-selective murine monoclonal antibody, SZ21, as well as human maternal anti-HPA-1a alloantisera. Successfully generation of mice whose platelets and endothelial cells express the human HPA-1a alloantigenic epitope will permit development of an authentic murine model of NAIT that should facilitate the investigation of a number of clinically-important issues in the diagnosis and treatment of platelet alloimmune disorders. Disclosures No relevant conflicts of interest to declare.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.