Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 µM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

Background data of 2-year-old male and female F344 gpt delta rats.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma.

Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas.

Establishment of the Global SEND Alliance (G-SEND) in Japan and efficient creation of electronic SEND datasets between CROs.

The Standard for Exchange of Nonclinical Data (SEND), adopted by the US Food and Drug Administration (FDA), is a set of regulations for digitalization and standardization of nonclinical study data; thus, related organizations have begun implementing processes in support of SEND. The Global Editorial and Steering Committee (GESC), which provides oversight of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), has prepared the SEND Controlled Terminology (CT) for toxicologic pathology. SEND provides electronic data standards created by the Clinical Data Interchange Standards Consortium (CDISC), and CDISC also collaborates in the implementation of SEND. Furthermore, the Pharmaceutical Users Software Exchange (PhUSE), which includes members of the US FDA, has conducted various activities to promote realistic and effective methods to implement SEND. As we reported in 2015, there is a significant variation in the efficiency and quality of SEND data implementation across pharmaceutical companies and contractors (CROs) globally. To address this problem, the Global SEND Alliance (G-SEND) was established in August 2018 to facilitate the coordination and standardization of SEND datasets across CROs in Asia. This paper reports the first method for organizationally and jointly creating consistent SEND datasets between CROs using G-SEND.

Cardiomyopathy Does Not Exacerbate the Severity of Pneumonia Caused by a SARS-CoV-2 Delta Variant in the J2N-k Hamster Model.

Cardiovascular disease is one of many risk factors that have been linked to increased severity or mortality in coronavirus disease 2019 (COVID-19) patients; however, the exact role of SARS-CoV-2 in the pathogenesis of cardiac inflammatory injury has not been established. A previous study reported that SARS-CoV-2 causes more severe disease with cardiomyopathy in a J2N-k animal model. Here, we investigated the sensitivity of J2N-k hamsters, as a cardiomyopathy animal model, to a delta strain of SARS-CoV-2 compared to J2N-n control animals. We found that J2N-k hamsters were less susceptible to this delta strain than J2N-n animals, and we found no evidence that cardiomyopathy is a risk factor in this animal model. Since the previous study reported that SARS-CoV-2 causes more severe disease with cardiomyopathy in the same animal model, further analysis of the relationship between cardiomyopathy and SARS-CoV-2 infection is needed.

DNA polymerase κ suppresses inflammation and inflammation-induced mutagenesis and carcinogenic potential in the colon of mice.

BACKGROUND: Chronic inflammation induces DNA damage and promotes cell proliferation, thereby increasing the risk of cancer. DNA polymerase κ (Pol κ), involved in translesion DNA synthesis, counteracts mutagenesis induced by inflammation in the colon of mice. In the present study, we examined whether Pol κ suppressed inflammation-induced colon tumorigenesis by treating inactivated Polk knock-in (Polk-/-) mice with dextran sulfate sodium (DSS), an inducer of colon inflammation. RESULTS: Male and female Polk-/- and Polk+/+ mice were administered 2% DSS in drinking water for six consecutive days, succeeded via a recovery period of 16 days, followed by 2% DSS for another two days. DSS treatment strongly induced colitis, and the severity of colitis was higher in Polk-/- mice than in Polk+/+ mice. The mice were sacrificed after 19 weeks from the initiation of the first DSS treatment and subjected to pathological examination and mutation analysis. DSS treatment induced colonic dysplasia, and the multiplicity of dysplasia was higher in Polk-/- mice than in Polk+/+mice. Some of the dysplasias in Polk-/- mice exhibited ß-catenin-stained nucleus and/or cytoplasm. Mutation frequencies in the gpt reporter gene were increased by DSS treatment in Polk-/- mice, and were higher than those in Polk+/+ mice. CONCLUSIONS: Pol κ suppresses inflammation and inflammation-induced dysplasia as well as inflammation-induced mutagenesis. The possible mechanisms by which Pol κ suppresses colitis- and colitis-induced dysplasia are discussed.

Constructing a continuous hemodiafiltration-type circulatory model of acute kidney injury in pigs.

INTRODUCTION: Animal-model experimental systems capable of reflecting the effects of devices for continuous renal replacement therapy (CRRT) on living organisms are limited; thus, aimed to construct an animal model of AKI-CRRT using pigs. METHODS: Pigs were subjected to renal artery ischemia-reperfusion injury (IRI) and then to a maximum of 24 h of continuous hemodiafiltration (CHDF)-type CRRT. RESULTS: Post-IRI, pigs' creatinine levels rose threefold, and they exhibited 24 h of anuria and clear aggravation of oxidative stress, demonstrating successful induction of AKI for CRRT. Post-CRRT, no significant changes in their vital signs or hematological parameters were observed. Creatinine and blood urea nitrogen clearance, as well as suppression of increases in oxidative stress, were also confirmed. CONCLUSION: We believe that the use of our model can enable the preclinical evaluation of the effects of under-development CRRT devices on living organisms under conditions similar to those encountered in an actual clinical setting.

New homozygous gpt delta transgenic rat strain improves an efficiency of the in vivo mutagenicity assay.

BACKGROUND: Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. RESULTS: The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi- deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. CONCLUSIONS: The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Improvement of a two-stage carcinogenesis model to detect modifying effects of endocrine disrupting chemicals on thyroid carcinogenesis in rats.

In order to improve the sensitivity of our previously established thyroid carcinogenesis model and to clarify whether endocrine disrupting chemicals with weak estrogenic activity have any modifying effects on the development of thyroid proliferative lesions, 6-week-old female castrated F344 rats were first given a single subcutaneous injection of 2000 mg/kg body weight of N-bis(2-hydroxypropyl)nitrosamine. From 1 week later, they received diets with: no supplement (basal diet (BD) group); cholesterol pellets containing 0.5 mg 17 beta-estradiol 3-benzoate (EB); or diet admixed with 1000 ppm methoxychlor (MXC) or 10,000 ppm bisphenol A (BPA) for 20 weeks. Furthermore, additional groups were administered 200 ppm sulfadimethoxine (SDM) in the drinking water simultaneously with the BD, EB, MXC or BPA treatments. Thyroid follicular cell hyperplasias, adenomas and/or carcinomas were induced only in the EB+SDM group, the incidences of non-malignant lesions being significantly increased, as compared with the BD+SDM group values. Furthermore, the serum level of thyroid stimulating hormone (TSH) was significantly increased in this group. No significant variation in quantitative values for thyroid proliferative lesions or TSH levels were observed in the other treated groups. The results of the present study convincingly indicate that EB, with strong estrogenic activity, but not MXC and BPA, with weak estrogenic activities, exerts promoting effects on thyroid carcinogenesis in rats. The present modified rat two-stage thyroid carcinogenesis model appears to have advantages over our previous model for screening purposes.

Gonadal toxicity of an ethanol extract of Psoralea corylifolia in a rat 90-day repeated dose study.

Ethanol extracts of seeds of Psoralea corylifolia are proposed as food additives for processed food preservation. An extract was administered by admixing into diet at concentrations of 0, 0.375, 0.75, 1.5 or 3.0% to 10 male and 10 female F344 rats each for 90 days to evaluate its toxicity. Body weight gain, food consumption and food conversion efficiency (body weight gain per food consumption) were lower in the extract-treated animals, except for the 0.375% males, as compared to the control animals. Absolute and/or relative testes weights in the 1.5 and 3.0% groups and those of ovaries in the 3.0% group were significantly (p < 0.01) lower than in the control group. On histopathological examination, seminiferous tubular atrophy and Leydig cell atrophy in the testes, and epithelial cell atrophy in the seminal vesicles and prostate were observed in the 1.5 and 3.0% males. Decrease in the number of corpora lutea associated with frequent necrotic follicles in the ovaries in the 1.5 and 3.0% females and less frequent endometrial glands in the uterus in the 3.0% females were also detected. These results might suggest disruption of the hypothalamus-pituitary-gonadal axis in Psoralea corylifolia-treated rats as possible mechanisms underlying this gonadal toxicity.

The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats.

Helper T (Th2) cells type 2 have a central role in host protective responses to gastrointestinal nematode parasite, Trichinella spiralis infection, but the actual effector mechanisms involved in parasite expulsion are still uncertain. Recent evidences suggest that mast cell recruitment and activation may associate with parasite elimination from host intestines in mice. On the other hand, IgE production may induce defensive responses to primary infection with the helminth in rats. The differences of host effector mechanisms to the same experimental infections might disturb our understanding of the host protective mechanisms to gastrointestinal nematode parasite infection. In order to redefine these differences, we investigated in detail the relationship between intestinal immune responses and worm expulsion following T. spiralis infection among several rat strains including mutants. As a result, there were significant correlations of parasite expulsion with mast cell hyperplasia in addition to serum IgE level. Moreover, mast cell-deficient and dysfunction rats showed delayed worm elimination from their gut. Therefore, the present study suggests that mast cells should also be one of the prominent effector cells involved in T. spiralis parasite expulsion in rats as well as mice.

Chromosomal mapping of host resistance loci to Trichinella spiralis nematode infection in rats.

The differences in host response among strains of rats to intestinal nematode parasite Trichinella spiralis infection could provide a powerful benefit for further elucidation of molecular interactions between the host and the parasite. Using several strains of rats, we previously observed that DA strain is a strong responder and F344 strain is a weak responder with respect to expulsion of the adult worm. To identify the host resistance loci, quantitative trait loci (QTLs) analysis in F2 population from crosses between DA and F344 strains was performed. One significant QTL (designated as Tspe) was mapped to the middle region of chromosome 9. In addition, the effect of DA allele at Tspe locus could act recessively and lead to the rejection of more adult worms from the gut. The results from the present study provide more insights on host-parasite interactions, which may be useful in facilitating the development of novel approaches for treatment and control of intestinal parasites in human and domestic livestock.

Lack of Carcinogenic Sensitivity of the Glandular Stomach in Heterozygous p53 Knockout Mice Given N-Methyl-N-Nitrosourea in their Drinking Water for 26 Weeks.

Gastric tumorigenic sensitivity to N-methyl-N-nitrosourea (MNU) was examined in heterozygous p53 knockout (p53(+/-)) CBA mice and their wild-type littermates (p53(+/+)). In Experiment 1, 37 male p53(+/-) or 38 male p53(+/+) CBA mice were given MNU in their drinking water at concentration of 50ppm (Group 1 or 4), 10ppm (group 2 or 5) or 0ppm (group3 or 6) for 26 weeks. In Experiment 2, p53(+/-) and p53(+/+) CBA mice of both sexes received water containing 50ppm MNU for 26 weeks. In Experiment 1, the incidences of hyperplasias in the glandular stomach observed in p53 (+/-) CBA mice treated with 50ppm and 10ppm MNU were significantly increased, as compared with the control group. No tumors were induced in the stomach of any treated groups. Some proliferative or non-neoplastic lesions were observed in some p53 (+/-) CBA mice, but there was no significant difference in their incidences between treated and control groups. In Experiment 2, the incidences of hyperplasias in the glandular stomach observed in p53 (+/-) CBA mice of both sexes treated with 50ppm MNU were not significantly increased, as compared with the treated p53(+/+) CBA group. One papilloma of the forestomach was observed only in a male p53(+/-) CBA mouse treated with 50ppm MNU. The present study suggests that p53 (+/-) CBA mice have low susceptibility to MNU-induced gastic carcinogenesis.

Enhancing effects of beta-estradiol 3-benzoate but not methoxychlor on the promotion/progression stage of chemically-induced mammary carcinogenesis in ovariectomized rats.

Modifying effects of beta-estradiol 3-benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg / kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion / progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.