Kazuto Yoshimi

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

Background Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR–Cas platform

The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Here we employ CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminate a single-nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 × DA)F1 hybrid embryos. Interestingly, the targeted allele, initially assessed by the allele-specific gRNA, is repaired by an interallelic gene conversion between homologous chromosomes. Using single-stranded oligodeoxynucleotides, we recover three recessive phenotypes: the albino phenotype by SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating animal models of human diseases and its potential use in gene therapy.

SsODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes

The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.

Enhanced colitis-associated colon carcinogenesis in a novel Apc mutant rat

To establish an efficient rat model for colitis‐associated colorectal cancer, azoxymethane and dextran sodium sulfate (AOM/DSS)‐induced colon carcinogenesis was applied to a novel adenomatous polyposis coli (Apc) mutant, the Kyoto Apc Delta (KAD) rat. The KAD rat was derived from ethylnitrosourea mutagenesis and harbors a nonsense mutation in the Apc gene (S2523X). The truncated APC of the KAD rat was deduced to lack part of the basic domain, an EB1‐binding domain, and a PDZ domain, but retained an intact β‐catenin binding region. KAD rats, homozygous for the Apc mutation on a genetic background of the F344 rat, showed no spontaneous tumors in the gastrointestinal tract. At 5 weeks of age, male KAD rats were given a single subcutaneous administration of AOM (20 mg/kg, bodyweight). One week later, they were given DSS (2% in drinking water) for 1 week. At week 15, the incidence and multiplicity of colon tumors developed in the KAD rat were remarkably severe compared with those in the F344 rat: 100 versus 50% in incidence and 10.7 ± 3.5 versus 0.8 ± 1.0 in multiplicity. KAD tumors were dominantly distributed in the rectum and distal colon, resembling human colorectal cancer. Accumulation of β‐catenin protein and frequent β‐catenin mutations were prominent features of KAD colon tumors. To our knowledge, AOM/DSS‐induced colon carcinogenesis using the KAD rat is the most efficient to induce colon tumors in the rat, and therefore would be available as an excellent model for human colitis‐associated CRC.

The utility of Apc-mutant rats in modeling human colon cancer.

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

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.

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

BackgroundChemotherapeutic 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.MethodsThe 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.ResultsIn 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.ConclusionThe 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.

Atopic dermatitis-like skin lesions with IgE hyperproduction and pruritus in KFRS4/Kyo rats

BACKGROUND Rats showing spontaneous atopic dermatitis (AD)-like skin lesions were observed in the Kyoto Fancy Rat Stock 4 (KFRS4) strain breeding colony. OBJECTIVE To establish the KFRS4 rat as a model of AD. METHODS The clinical symptoms of AD-like skin lesions were assessed by scoring the degree of dermatitis and examining scratching behavior. The transepidermal water loss was measured to evaluate skin barrier function. Cells infiltrating the skin lesions were identified using histological and immunohistological analyses. IgE and cytokine levels were measured to examine immune status. An ointment treatment experiment was carried out to characterize dermatitis in the KFRS4 rats. RESULTS Dermatitis initially appeared around 4 months of age and rapidly worsened from 6 to 8 months of age. The skin lesions accompanied scratching behavior and were predominantly observed in females. The increased transepidermal water loss indicated skin barrier dysfunction. Extensive infiltration of eosinophils, mast cells and lymphocytes was observed in the skin lesions. The plasma IgE level increased in accord with increasing severity of dermatitis. The Th2 and Th17 cytokine mRNA levels were significantly higher in the skin-draining lymph nodes than those in the non-skin-draining lymph nodes. It was demonstrated that betamethasone improved the symptoms of dermatitis. These findings demonstrated that dermatitis in the KFRS4 rats closely resembled that seen in human AD. CONCLUSION Female KFRS4 rats have the potential to serve as an animal model of human AD.

Utility of Apc-mutant rats with a colitis-associated colon carcinogenesis model for chemoprevention studies.

Establishment of an efficient rat model for colitis-associated colon carcinogenesis is critical for evaluation of the potency of cancer-preventive agents on carcinogenesis. In the present study, we examined whether the Kyoto Apc Delta (KAD) rat, a novel adenomatous polyposis coli mutant rat strain, is useful for detection of potential chemopreventive agents when this rat is used for azoxymethane (AOM) plus dextran sulfate sodium (DSS)-induced colitis-associated colon carcinogenesis with well-known cancer chemopreventive agents, such as celecoxib and (−)-epigallocatechin-3-gallate (EGCG). Male KAD rats were administered a single subcutaneous injection of AOM (20 mg/kg body weight) at 5 weeks of age and 2% DSS in their drinking water for subsequent 7 days starting at 1 week after the AOM injection. The rats were also treated with either celecoxib (500 ppm in the diet) or EGCG (0.1% in their drinking water), and the effects of these agents on the development of colonic tumors were examined. At sacrifice (19 weeks of age), treatment with both celecoxib (74% inhibition, P<0.01) and EGCG (71% inhibition, P<0.05) significantly inhibited the development of colitis-associated colon tumors. Serum levels of oxidative stress products, which were increased by AOM/DSS treatment, were decreased by celecoxib and EGCG. Administration of these agents moreover lowered the serum levels of inflammatory mediators, including tumor necrosis factor-&agr;, interleukin-6, cyclooxygenase-2, and inducible nitric oxide synthase, in the AOM/DSS-treated KAD rats. These findings suggest that this AOM/DSS-induced colon carcinogenesis model using KAD rats may be useful for evaluation of the efficacy of cancer-preventive agents.

CLICK: one-step generation of conditional knockout mice

BackgroundCRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.ResultsHere, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.ConclusionsThe high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.