Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

Characterization of novel, severely immunodeficient PrkdcΔex57/Δex57 mice.

Severely immunodeficient mice are useful for understanding the pathogenesis of certain tumors and for developing therapeutic agents for such tumors. In addition, engraftment of these mice with human hematopoietic cells can yield information that helps us understand the in vivo molecular mechanisms underlying actual human viral infections. In our present research, we discovered a novel, severely immunodeficient strain of mice having a mutation in exon 57 of the Prkdc gene (PrkdcΔex57/Δex57) in an inbred colony of B10.S/SgSlc mice. Those PrkdcΔex57/Δex57 mice showed thymic hypoplasia and lack of mature T cells and B cells in peripheral lymphoid tissues, resulting in very low levels of production of serum immunoglobulins. In addition, those mice were highly susceptible to influenza viruses due to the lack of acquired immune cells. On the other hand, since they had sufficient numbers of NK cells, they rejected tumor transplants, similarly to Prkdc+/+ mice. Next, we generated Foxn1nu/nuPrkdcΔex57/Δex57Il2rg-/- (NPG) mice on the BALB/cSlc background, which lack all lymphocytes such as T cells, B cells and innate lymphoid cells, including NK cells. As expected, these mice were able to undergo engraftment of human tumor cell lines. These findings suggest that PrkdcΔex57/Δex57 mice will be useful as a novel model of immunodeficiency, while NPG mice will be useful for xenografting of various malignancies.

Down-Regulation of Astrocytic Kir4.1 Channels during the Audiogenic Epileptogenesis in Leucine-Rich Glioma-Inactivated 1 (Lgi1) Mutant Rats.

The dysfunction of astrocytic inwardly rectifying potassium (Kir) 4.1 channels, which mediate the spatial potassium-buffering function of astrocytes, is known to be involved in the development of epilepsy. Here, we analyzed the Kir4.1 expressional changes in Leucine-Rich Glioma-Inactivated 1 (Lgi1) mutant rats, which is a model of autosomal dominant lateral temporal lobe epilepsy in humans, to clarify the role of astrocytic Kir4.1 channels in Lgi1-related epileptogenesis. Priming acoustic stimulation (at postnatal day 16) conferred seizure susceptibility on Lgi1 mutant rats, which evoked audiogenic seizures with test stimulation at eight weeks. In the seizure-susceptible Lgi1 mutant rats (before test stimulation), astrocytic Kir4.1 expression was down-regulated region-specifically in the cerebral cortex, hippocampus, and amygdala. In addition, prophylactic treatments of Lgi1 mutant rats with valproic acid (VPA, 30 mg/kg and 200 mg/kg) for two weeks prevented both the development of seizure susceptibility and the down-regulation of Kir4.1 expression in astrocytes. The present study demonstrated for the first time that the astrocytic Kir4.1 expression was reduced in the Lgi1-related seizure model, suggesting that the down-regulation of Kir4.1 channels in astrocytes is involved in audiogenic epileptogenesis caused by Lgi1 mutation. In addition, VPA seemed to have a prophylactic effect on Lgi1-related seizures.

Seipin is necessary for normal brain development and spermatogenesis in addition to adipogenesis.

Seipin, encoded by BSCL2 gene, is a protein whose physiological functions remain unclear. Mutations of BSCL2 cause the most-severe form of congenital generalized lipodystrophy (CGL). BSCL2 mRNA is highly expressed in the brain and testis in addition to the adipose tissue in human, suggesting physiological roles of seipin in non-adipose tissues. Since we found BSCL2 mRNA expression pattern among organs in rat is similar to human while it is not highly expressed in mouse brain, we generated a Bscl2/seipin knockout (SKO) rat using the method with ENU (N-ethyl-N-nitrosourea) mutagenesis. SKO rats showed total lack of white adipose tissues including mechanical fat such as bone marrow and retro-orbital fats, while physiologically functional brown adipose tissue was preserved. Besides the lipodystrophic phenotypes, SKO rats showed impairment of spatial working memory with brain weight reduction and infertility with azoospermia. We confirmed reduction of brain volume and number of sperm in human patients with BSCL2 mutation. This is the first report demonstrating that seipin is necessary for normal brain development and spermatogenesis in addition to white adipose tissue development.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

The VF rat with abnormal myelinogenesis has a mutation in Dopey1.

The vacuole formation (VF) rat is an autosomal recessive myelin mutant characterized by generalized tremor, hypomyelination, and periaxonal vacuole formation of the central nervous system (CNS). Here, we report the most likely causative gene for neurological disease in the VF rat and pursue its roles in the development and maintenance of the CNS myelin. We identified a nonsense mutation in the dopey family member 1 (Dopey1) located on rat chromosome 8. Expression level of Dopey1 mRNA was decreased and DOPEY1 protein was undetectable both in the white and gray matter of the spinal cords in the VF rats. Double immunohistochemistry demonstrated that DOPEY1 was mainly expressed in neurons and oligodendrocytes in the wild-type rats, whereas no positive cells were detected in the VF rats. We also demonstrated a marked reduction in myelin components both at mRNA and protein levels during myelinogenesis in the VF rats. In addition, proteolipid protein and myelin-associated glycoprotein accumulated in oligodendrocyte cell body, suggesting that Dopey1 is likely to be involved in the traffic of myelin components. Our results highlighted the importance of Dopey1 for the development and maintenance of the CNS myelin.

A rat model for LGI1-related epilepsies.

Mutations of the leucine-rich glioma-inactivated 1 (LGI1) gene cause an autosomal dominant partial epilepsy with auditory features also known as autosomal-dominant lateral temporal lobe epilepsy. LGI1 is also the main antigen present in sera and cerebrospinal fluids of patients with limbic encephalitis and seizures, highlighting its importance in a spectrum of epileptic disorders. LGI1 encodes a neuronal secreted protein, whose brain function is still poorly understood. Here, we generated, by ENU (N-ethyl-N-nitrosourea) mutagenesis, Lgi1-mutant rats carrying a missense mutation (L385R). We found that the L385R mutation prevents the secretion of Lgi1 protein by COS7 transfected cells. However, the L385R-Lgi1 protein was found at low levels in the brains and cultured neurons of Lgi1-mutant rats, suggesting that mutant protein may be destabilized in vivo. Studies on the behavioral phenotype and intracranial electroencephalographic signals from Lgi1-mutant rats recalled several features of the human genetic disorder. We show that homozygous Lgi1-mutant rats (Lgi1(L385R/L385R)) generated early-onset spontaneous epileptic seizures from P10 and died prematurely. Heterozygous Lgi1-mutant rats (Lgi1(+/L385R)) were more susceptible to sound-induced, generalized tonic-clonic seizures than control rats. Audiogenic seizures were suppressed by antiepileptic drugs such as carbamazepine, phenytoin and levetiracetam, which are commonly used to treat partial seizures, but not by the prototypic absence seizure drug, ethosuximide. Our findings provide the first rat model with a missense mutation in Lgi1 gene, an original model complementary to knockout mice. This study revealed that LGI1 disease-causing missense mutations might cause a depletion of the protein in neurons, and not only a failure of Lgi1 secretion.

Tumor suppressor APC protein is essential in mucosal repair from colonic inflammation through angiogenesis.

Mucosal repair after acute colonic inflammation is central to maintaining mucosal homeostasis. Failure of mucosal repair often leads to chronic inflammation, sometimes associated with inflammatory bowel disease (IBD). The adenomatous polyposis coli (APC) tumor suppressor gene regulates the Wnt signaling pathway, which is essential for epithelial development, and inactivation of APC facilitates colorectal cancer. Our previous study suggested that APC is involved in pathogenesis of colonic inflammation; however, its role in mucosal repair remains unknown. In this article, we report that colitis induced by dextran sodium sulfate persisted with delayed mucosal repair in Kyoto Apc Delta (KAD) rats lacking the APC C terminus. Defects in the repair process were accompanied by an absence of a fibrin layer covering damaged mucosa and reduced microvessel angiogenesis. APC was up-regulated in vascular endothelial cells (VECs) in inflamed mucosa in KAD and F344 (control) rats. The VECs of KAD rats revealed elevated cell adhesion and low-branched and short-length tube formation. We also found that DLG5, which is associated with IBD pathogenesis, was up-regulated in VECs in inflamed mucosa and interacted with the C terminus of APC. This finding suggests that loss of interaction between the APC C terminus and DLG5 affects VEC morphology and function and leads to persistence of colitis. Therefore, APC is essential for maintenance of intestinal mucosal homeostasis and can consequently contribute to IBD pathogenesis.

A mutation in the gene encoding mitochondrial Mg²+ channel MRS2 results in demyelination in the rat.

The rat demyelination (dmy) mutation serves as a unique model system to investigate the maintenance of myelin, because it provokes severe myelin breakdown in the central nervous system (CNS) after normal postnatal completion of myelination. Here, we report the molecular characterization of this mutation and discuss the possible pathomechanisms underlying demyelination. By positional cloning, we found that a G-to-A transition, 177 bp downstream of exon 3 of the Mrs2 (MRS2 magnesium homeostasis factor (Saccharomyces cerevisiae)) gene, generated a novel splice acceptor site which resulted in functional inactivation of the mutant allele. Transgenic rescue with wild-type Mrs2-cDNA validated our findings. Mrs2 encodes an essential component of the major Mg²+ influx system in mitochondria of yeast as well as human cells. We showed that the dmy/dmy rats have major mitochondrial deficits with a markedly elevated lactic acid concentration in the cerebrospinal fluid, a 60% reduction in ATP, and increased numbers of mitochondria in the swollen cytoplasm of oligodendrocytes. MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was dominantly expressed in neurons rather than oligodendrocytes and was ultrastructurally observed in the inner membrane of mitochondria. Our observations led to the conclusion that dmy/dmy rats suffer from a mitochondrial disease and that the maintenance of myelin has a different mechanism from its initial production. They also established that Mg²+ homeostasis in CNS mitochondria is essential for the maintenance of myelin.

A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.

Generation of leptin-deficient Lepmkyo/Lepmkyo rats and identification of leptin-responsive genes in the liver.

Leptin is one of the key molecules in maintaining energy homeostasis. Although genetically leptin-deficient Lep(ob)/Lep(ob) mice have greatly contributed to elucidating leptin physiology, the use of more than one species can improve the accuracy of analysis results. Using the N-ethyl-N-nitrosourea mutagenesis method, we generated a leptin-deficient Lep(mkyo)/Lep(mkyo) rat that had a nonsense mutation (Q92X) in leptin gene. Lep(mkyo)/Lep(mkyo) rats showed obese phenotypes including severe fatty liver, which were comparable to Lep(ob)/Lep(ob) mice. To identify genes that respond to leptin in the liver, we performed microarray analysis with Lep(mkyo)/Lep(mkyo) rats and Lep(ob)/Lep(ob) mice. We sorted out genes whose expression levels in the liver of Lep(mkyo)/Lep(mkyo) rats were changed from wild-type (WT) rats and were reversed toward WT rats by leptin administration. In this analysis, livers were sampled for 6 h, a relatively short time after leptin administration to avoid the secondary effect of metabolic changes such as improvement of fatty liver. We did the same procedure in Lep(ob)/Lep(ob) mice and selected genes whose expression patterns were common in rat and mouse. We verified their gene expressions by real-time quantitative PCR. Finally, we identified eight genes that primarily respond to leptin in the liver commonly in rat and mouse. These genes might be important for the effect of leptin in the liver.

Role of novel rat-specific Fc receptor in macrophage activation associated with crescentic glomerulonephritis.

Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain.

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.

Cblb is a major susceptibility gene for rat type 1 diabetes mellitus.

The autoimmune disease type 1 diabetes mellitus (insulin-dependent diabetes mellitus, IDDM) has a multifactorial etiology. So far, the major histocompatibility complex (MHC) is the only major susceptibility locus that has been identified for this disease and its animal models. The Komeda diabetes-prone (KDP) rat is a spontaneous animal model of human type 1 diabetes in which the major susceptibility locus Iddm/kdp1 accounts, in combination with MHC, for most of the genetic predisposition to diabetes. Here we report the positional cloning of Iddm/kdp1 and identify a nonsense mutation in Cblb, a member of the Cbl/Sli family of ubiquitin-protein ligases. Lymphocytes of the KDP rat infiltrate into pancreatic islets and several tissues including thyroid gland and kidney, indicating autoimmunity. Similar findings in Cblb-deficient mice are caused by enhanced T-cell activation. Transgenic complementation with wildtype Cblb significantly suppresses development of the KDP phenotype. Thus, Cblb functions as a negative regulator of autoimmunity and Cblb is a major susceptibility gene for type 1 diabetes in the rat. Impairment of the Cblb signaling pathway may contribute to human autoimmune diseases, including type 1 diabetes.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

Imbalance of glutamatergic and GABAergic neurotransmission in audiogenic seizure-susceptible Leucine-rich glioma-inactivated 1 (Lgi1)-mutant rats.

Leucine-rich glioma-inactivated 1 (LGI1) was identified as a causative gene of autosomal dominant lateral temporal lobe epilepsy. We previously reported that Lgi1-mutant rats carrying a missense mutation (L385R) showed audiogenic seizure-susceptibility. To explore the pathophysiological mechanisms underlying Lgi1-related epilepsy, we evaluated changes in glutamate and GABA release in Lgi1-mutant rats. Acoustic priming (AP) for audiogenic seizure-susceptibility was performed by applying intense sound stimulation (130 dB, 10 kHz, 5 min) on postnatal day 16. Extracellular glutamate and GABA levels in the hippocampus CA1 region were evaluated at 8 weeks of age, using in vivo microdialysis techniques. Under naïve conditions without AP, glutamate and GABA release evoked by high-K+ depolarization was more prominent in Lgi1-mutant than in wild-type (WT) rats. The AP treatment on day 16 significantly increased basal glutamate levels and depolarization-induced glutamate release both in Lgi1-mutant and WT rats, yielding greater depolarization-induced glutamate release in Lgi1-mutant rats. On the other hand, the AP treatment enhanced depolarization-induced GABA release only in WT rats, and not in Lgi1-mutant rats, illustrating reduced GABAergic neurotransmission in primed Lgi1-mutant rats. The present results suggest that enhanced glutamatergic and reduced GABAergic neurotransmission are involved in the audiogenic seizure-susceptibility associated with Lgi1-mutation.

Hypercholesterolemia and atherosclerosis in low density lipoprotein receptor mutant rats.

To establish low density lipoprotein receptor (LDLR) mutant rats as a hypercholesterolemia and atherosclerosis model, we screened the rat LDLR gene for mutations using an N-ethyl-N-nitrosourea mutagenesis archive of rat gene data, and identified five mutations in its introns and one missense mutation (478T>A) in exon 4. The C160S mutation was located in the ligand binding domain of LDLR and was revealed to be equivalent to mutations (C160Y/G) identified in human familial hypercholesterolemia (FH) patients. The wild type, heterozygous, and homozygous mutant rats were fed a normal chow diet or a high fat high cholesterol (HFHC) diet from the age of 10 weeks for 16 weeks. The LDLR homozygous mutants fed the normal chow diet showed higher levels of plasma total cholesterol and LDL cholesterol than the wild type rats. When fed the HFHC diet, the homozygous mutant rats exhibited severe hyperlipidemia and significant lipid deposition from the aortic arch to the abdominal aorta as well as in the aortic valves. Furthermore, the female homozygous mutants also developed xanthomatosis in their paws. In conclusion, we suggest that LDLR mutant rats are a useful novel animal model of hypercholesterolemia and atherosclerosis.

Use of a chemically induced-colon carcinogenesis-prone Apc-mutant rat in a chemotherapeutic bioassay.

BACKGROUND: Chemotherapeutic bioassay for colorectal cancer (CRC) with a rat model bearing chemically-induced CRCs plays an important role in the development of new anti-tumor drugs and regimens. Although several protocols to induce CRCs have been developed, the incidence and number of CRCs are not much enough for the efficient bioassay. Recently, we established the very efficient system to induce CRCs with a chemically induced-colon carcinogenesis-prone Apc-mutant rat, Kyoto Apc Delta (KAD) rat. Here, we applied the KAD rat to the chemotherapeutic bioassay for CRC and showed the utility of the KAD rat. METHODS: The KAD rat has been developed by the ENU mutagenesis and carries a homozygous nonsense mutation in the Apc gene (S2523X). Male KAD rats were given a single subcutaneous injection of AOM (20 mg/kg body weight) at 5 weeks of age. Starting at 1 week after the AOM injection, they were given 2% DSS in drinking water for 7 days. Tumor-bearing KAD rats were divided into experimental and control groups on the basis of the number of tumors observed by endoscopy at week 8. The 5-fluorouracil (5-FU) was administrated intravenously a dose of 50 or 75 mg/kg weekly at week 9, 10, and 11. After one-week interval, the 5-FU was given again at week 13, 14, and 15. At week 16, animals were sacrificed and tumor number and volume were measured macroscopically and microscopically. RESULTS: In total 48 tumors were observed in 27 KAD rats with a 100% incidence at week 8. The maximum tolerated dose for the KAD rat was 50 mg/kg of 5-FU. Macroscopically, the number or volume of tumors in the 5-FU treated rats was not significantly different from the control. Microscopically, the number of adenocarcinoma in the 5-FU treated rats was not significantly different (p < 0.02) from that of the control. However, the volume of adenocarcinomas was significantly lower than in the control. Anticancer effect of the 5-FU could be obtained only after the 16 weeks of experimental period. CONCLUSION: The use of the AOM/DSS-treated tumor-bearing KAD rats could shorten the experimental period and reduce the number of animals examined in the chemotherapeutic bioassay. The efficient bioassay with the AOM/DSS-treated tumor-bearing KAD rats would promote the development of new anti-tumor drugs and regimens.

Long-term preservation of freeze-dried mouse spermatozoa.

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.