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News Article | May 10, 2017
Site: www.nature.com

To generate CRISPR–Cas9 plasmids targeting the last exon of LGR5 (exon 18) or KRT20 (exon 8), 20-bp target sequences were cloned into a pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene 42230) to obtain single vectors bicistronically expressing sgRNA and human codon-optimized Cas9 nuclease as previously described36. The sgRNA sequences targeting LGR5 or KRT20 are available in Supplementary Table 1. To construct donor vectors for LGR5–GFP- and KRT20–GFP-knock-in, 5′ and 3′ homology arms (1 kbp each) were amplified by PCR and cloned into an Ires-GFP-loxp-pEF1α-RFP-T2A-puro-loxp plasmid (HR180PA-1, SBI) using the In-Fusion HD Cloning kit (Clontech). For CreER or iCaspase9-T2A-tdTomato knock-in, we replaced GFP of the LGR5–GFP or KRT20–GFP construct with CreER or iCaspase9-T2A-tdTomato, respectivley. The final plasmid sequences were verified by DNA sequencing. To obtain a rainbow reporter, the rainbow cassette was excised from a CMV-Brainbow-2.1R plasmid (Addgene 18723) and cloned into a PiggyBac vector (PB510B-1, SBI). For bioluminescent imaging, we cloned optimized firefly luciferase luc2 into a GFP-expressing PiggyBac vector (PB513B-1, SBI). Knock-in efficiency and diver mutation profiles for each CCO line are available in Supplementary Table 2. All organoids were established as previously reported16 from patients who had given informed consent under the ethical committee of Keio University School of Medicine. The organoids were embedded in Matrigel and cultured with previously described basal culture medium37, specifically Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1× B27 (Life Technologies), 10 nM gastrin I (Sigma) and 1 mM N-acetylcysteine (Sigma). The following niche factors were added to the basal culture medium depending on the niche requirements of CRC organoid lines: 50 ng ml−1 mouse recombinant EGF, 100 ng ml−1 mouse recombinant noggin (PeproTech) and 500 nM A83-01 (Tocris). We electroporated the vectors under previously reported conditions37. Three days after electroporation, the organoids were selected with puromycin (2 μg ml−1) treatment for two days. For in vitro ablation experiments, we treated the organoids with 1 nM dimerizer (AP20187, Clontech). Drug-resistant organoid clones were manually selected and expanded individually. Genomic DNA was isolated using the QIAamp DNA blood mini kit (Qiagen). Legitimate knock-in was determined by PCR. Southern blotting was performed based on the standard procedure using 1 μg of genomic DNA. The sequences of PCR primers and Southern blot probes are shown in Supplementary Table 1. The puromycin-RFP selection cassette flanked by loxP sequences was excised by transient infection of Cre-expressing adenovirus (TaKaRa) at multiplicity of infection of 5–10. After the infection, we manually selected and cloned RFP− organoids. Deletion of the puromycin cassette was validated by PCR diagnostics. Percentage of successful knock-in for each line is shown in Supplementary Table 2. Once a knock-in reporter CCO was cloned, we used the same clone for further experiments. Organoids were dissociated into single cells with TrypLE Express (Life Technology), and large clusters were removed with a CellTrics 20-μm cell strainer (Partec). The cells were washed with cold PBS and stained with 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences) to exclude dead cells. Single cells were gated based on the SSC-H versus SSC-W profile. The cells were subsequently analysed using a flow cytometer with a 70-μm nozzle (FACS JAZZ, BD Biosciences). Then, 1,000 sorted cells were embedded in 25 μl of Matrigel and cultured in a 48-well plate for 7–10 days. We added 10 μM Y27632 for the first two days of culture, and the organoid colony formation was assessed using a BZX-700 fluorescence microscope (Keyence). RNA was extracted from 1 × 106 sorted cells using the RNeasy Plus mini kit (Qiagen). The RNA quality was determined by the RNA integrity number (RIN) value with the RNA6000 assay (Agilent). Only specimens with RIN > 7.0 were used in this study. Gene expression was determined by microarray (GeneChip PrimeView Human Gene Expression Array, Affymetrix) according to the manufacturer’s instructions. The data were normalized using the robust multi-array analysis implemented in the R package affy. The probes were summarized into genes by selecting probes with the highest median absolute deviation value per gene. GSEA was performed using gsea (in the R package phenoTest) with 1,000 permutations. Two independent intestinal stem cell signature gene sets from refs 17, 38 were used. All animal procedures were approved by the Keio University School of Medicine Animal Care Committee. NOD/Shi-scid,IL-2Rγnull (NOG) mice39 (7–12 weeks of age, male) were obtained from the Central Institute for Experimental Animals (CIEA, Japan). Organoids with the indicated genetic reporter and with or without GFP-luc2-reporter, equivalent to 1 × 105 cells, were xenotransplanted subcutaneously or into the renal subcapsules as previously described40. We monitored the tumour size with a calliper or through bioluminescence imaging. Tumour volumes were measured according to the formula (length × width2) / 2. Once any individual tumour reached 2 cm in size, the mouse was euthanized. For bioluminescence imaging, we intraperitoneally administered 3 mg of D-luciferin (SPI, Tokyo) to tumour-bearing mice 10–20 min before imaging and anaesthetized the animals with isoflurane. The bioluminescence signal was measured with an IVIS imaging system (Xenogen), and the specific signal was calculated as the ratio of photon counts from the region of interest to counts from a background region. The grafts were fixed for subsequent histological analyses. An investigator blinded to the experimental conditions measured the tumour sizes. For the lineage-tracing experiments, each mouse received a single intraperitoneal injection of 0.25 mg (clonal dose) or 1 mg of tamoxifen (Sigma-Aldrich) diluted in corn oil. For the ablation studies, 40 μg of dimerizer was administered for five days daily for short term ablation and on alternate days for long term ablation. To label the proliferating cells, we intraperitoneally administered BrdU (40 mg kg−1, BD Biosciences) and EdU (10 mg kg−1, Life Technologies) at the indicated times. For chemotherapeutic studies, CTX (40 mg kg−1, Merck Serono) or oxaliplatin (15 mg kg−1, AdooQ Bioscience) was administered intraperitoneally at the indicated times. We isolated tumours from xenografted mice and immediately fixed them with 4% paraformaldehyde. Eight-micrometre OCT frozen tissue sections or 5-μm paraffin-embedded tissue sections were processed using a standard histological protocol. For rainbow fluorescent imaging, the frozen sections were visualized using an SP8 confocal microscope (Leica) with the following settings: mCFP was excited at 405 nm and collected using a 480–485-nm filter, nuclear GFP was excited at 488 nm and collected using a 494–507-nm filter, EYFP was excited at 514 nm and collected using a 560–566-nm filter, and RFP was excited at 552 nm and collected using a 601–665-nm filter. Nuclei were counterstained with the near-infrared nuclear dye DRAQ5 (BioStatus). For DLS 3D imaging, the whole tumours were cut into 1–2 mm3 pieces, fixed and embedded in agarose gel. 3D images were acquired using the Leica SP8 DLS system. The proportion of surviving clones was determined by counting the number of RFP+ cells at day 3 and day 31 after tamoxifen administration. Clone identification and raw volume measurement were carried out automatically using the ImageJ 3D-image processing package ‘3D object counter’41, 42. False identification rate of this automatic measurement was determined manually by random sampling. Raw volume was adjusted by randomly subtracting a proportion of clones according to the false-rate. The threshold volume for total colonies on day 3 was set as <2 × 105 μm3 and for large colonies on day 31 as >2 × 105 μm3 (equivalent to 20 cells). Colony-formation efficiency was defined as the ratio of the number of large colonies on day 31 to the number of clones on day 3. For immunostaining, the following primary antibodies were used: mouse anti-cytokeratin-20 (M7019, clone K 20.8, Dako, 1:50), goat anti-GFP (ab6673, Abcam, 1:200), rabbit anti-Ki67 (ab16667, Abcam, 1:100), mouse anti-α smooth muscle actin ab-1 (MS-113-P, Thermo Scientific, 1:800), mouse anti-BrdU (347580, BD, 1:100), anti-cleaved caspase-3 (9661, Cell Signaling, 1:100) and anti-tdTomato (600-401-379, ROCKLAND, 1:500). Alexa Fluor 488-, 568- or 647-conjugated secondary antibodies (Life Technologies, donkey anti-mouse, rabbit, rat or goat antibodies) were used at 1:200 dilution. For EdU staining, we used the Click-IT Plus EdU Imaging kit (Life Technologies) according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 or DAPI. Images were captured with a Leica SP8 confocal microscope or a BZX-700 fluorescence microscope (Keyence). To count the number of BrdU/EdU-stained cells, we used Imaris (Bitplane). In situ hybridization was performed using an RNAscope 2.5HD kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. For each experiment, we used PPIB and DapB genes as positive and negative controls, respectively. Tumour tissues were homogenized using TissueLyser LT (Qiagen) and RNA was extracted with the RNAeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the Omniscript RT kit (Qiagen). Quantitative real-time PCR was performed on LightCycler 96 (Roche Diagnostics) using FastStart Essential DNA Probes Master (Roche Diagnostics) and the cDNAs as templates. Relative LGR5 expression to ACTB was calculated based on the comparative C method. Primers and probes for LGR5 and ACTB are available in Supplementary Table 1. The sample size was determined by previous experience and preliminary experiments. The vehicle/dimerizer/chemotherapy-treated group was randomly assigned on the basis of tumour size at the time of injection. Appropriate statistical analyses were performed dependent on the comparisons referenced in the figure legends. The n values represent biological replicates. All graphs show mean and error bars represent the standard error of the mean (s.e.m.). Genetic mutation data of organoids are summarized in Supplementary Table 2 and described in ref. 16. The microarray dataset generated in this study is available in the Gene Expression Omnibus (accession number: GSE83513). All other data are available from the corresponding author upon reasonable request.

News Article | December 25, 2016
Site: www.prweb.com

Researchers affiliated with the Kawasaki INnovation Gateway at SKYFRONT, have successfully generated the first ever non-human primate X-SCID models by using two genome editing techniques. The findings were published in Cell Stem Cell, July 2016. Further information about science and technology projects at Kawasaki City is available in the Kawasaki SkyFront iNewsletter that highlights research being conducted by scientists and industries affiliated with Kawasaki INnovation Gateway at SKYFRONT (KING SKYFRONT)—the City’s flagship science and technology hub launched in 2013 to focus on open innovation in the life sciences and environment. First non-human primate model for severe combined immunodeficiency: While many insights are gained into diseases and genetic disorders from rodent models, there is a pressing need to find models that can more accurately represent disease progression in the human body, such as non-human primates. The latest advances in genome editing technology are opening doors to generating non-human primate models for studying specific genetic disorders, such as X-linked severe combined immunodeficiency (X-SCID). X-SCID patients do not produce enough T-cells and natural killer cells to tackle infections. The condition stems from a defective gene, IL2-RG, and scientists are keen to know more about the disorder and how to improve treatment. Now, Erika Sasaki at the Central Institute for Experimental Animals, Kawasaki, and co-workers across Japan and the US, have successfully generated the first ever non-human primate X-SCID models by using two genome editing techniques – zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The team worked with marmoset monkeys because they are easy to handle and have a small body size perfect for testing small amounts of new drugs in preclinical trials. They screened the most effective ZFNs to knock-out IL2-RG gene among the 16 pairs of candidate ZFNs cultured cells. They then injected the selected ZFNs into 1 cell stage marmoset embryos and performed a specialized screening test to check for, and avoid, mosaicism (if the models carrying the mutant gene in all of the tissues in the body or not). The researchers then transferred the embryos to surrogate mothers. Of the 5 ZFN and 4 TALEN baby marmosets that were born, three have since survived to adulthood and represent the first group of X-SCID non-human primate models ever created. Further tests showed that the IL2-RG knockout marmoset models exhibited strong phenotypic similarities to human patients with X-SCID. Sasaki’s team are confident that their work paves the way for the creation of multiple gene knock-out models for specific diseases and genetic disorders. Reference and affiliations K. Sato1, R. Oiwa1, W. Kumita1, R. Henry2, T. Sakuma3, R. Ito1, R. Nozu1, T. Inoue1, I. Katano1, K. Sato4, N. Okahara1, J. Okahara1, Y. Shimizu1, M. Yamamoto1, K. Hanazawa5, T. Kawakami6, Y. Kametani7, R. Suzuki8, T. Takahashi1, E. J. Weinstein2, T. Yamamoto3, Y. Sakakibara4, S. Habu9, J. Hata1, H. Okano10*, & E. Sasaki1,11,* Generation of a nonhuman primate model of severe combined immunodeficiency using highly efficient genome editing. Cell Stem Cell 19 127-138 (2016) DOI: http://dx.doi.org/10.1016/j.stem.2016.06.003 1. Central Institute for Experimental Animals, Kawasaki, Kanagawa 210-0821, Japan 2. Horizon Discovery, Saint Louis, MO 63146, USA 3. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan 4. Department of Biosciences and Informatics, Keio University, Yokohama, Kanagawa 223-8522, Japan 5. Department of Oncology, Juntendo University Nerima Hospital, Nerima-ku, Tokyo 177-8521, Japan 6. Medical Proteo Scope Company, Ltd., Yokohama, Kanagawa 236-0004, Japan 7. Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan 8. Department of Rheumatology and Clinical Immunology, Clinical Research Center for Rheumatology and Allergy, Sagamihara National Hospital, Sagamihara, Kanagawa 252-0392, Japan 9. Atopy Research Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan 10. Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan 11. Advanced Research Center, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan About KING SKYFRONT KING SKYFRONT is located on the opposite side of the Tama River that separates Tokyo International Airport (also known as Haneda Airport) and the Tonomachi district of Kawasaki. The Airport plays an important role in the globalization of the innovative activities of scholars, industrialists and City administrators based at KING SKYFRONT. KING SKYFRONT was launched in 2013 as a base for scholars, industrialists and government administrators to work together to devise real life solutions to global issues in the life sciences and environment.

Okano H.,Keio University | Hikishima K.,Keio University | Hikishima K.,Central Institute for Experimental Animals | Iriki A.,RIKEN | Sasaki E.,Central Institute for Experimental Animals
Seminars in Fetal and Neonatal Medicine | Year: 2012

The common marmoset (Callithrix jacchus), a small New World primate, has been attracting much attention in the research field of biomedical science and neuroscience, based on its (i) cross-reactivity with human cytokines or hormones, (ii) comparative ease in handling due to its small size, (iii) high reproductive efficiency, (iv) establishment of basic research tools, and (v) advantages of its unique behavioral and cognitive characters. Various neurological disease models have been developed in the common marmoset, including Parkinson's disease, Huntington's disease, Alzheimer's disease, stroke, multiple sclerosis and spinal cord injury. We recently developed transgenic common marmoset with germline transmission, which is expected to provide a new animal model for the study of human diseases. In this review, we summarize the recent progress of biomedical research and neuroscience using common marmoset as an excellent model system. © 2012 Elsevier Ltd.

Sasaki E.,Keio University | Sasaki E.,Central Institute for Experimental Animals
Neuroscience Research | Year: 2015

Genetically modified mice have contributed much to studies in the life sciences. In some research fields, however, mouse models are insufficient for analyzing the molecular mechanisms of pathology or as disease models. Often, genetically modified non-human primate (NHP) models are desired, as they are more similar to human physiology, morphology, and anatomy. Recent progress in studies of the reproductive biology in NHPs has enabled the introduction of exogenous genes into NHP genomes or the alteration of endogenous NHP genes. This review summarizes recent progress in the production of genetically modified NHPs, including the common marmoset, and future perspectives for realizing genetically modified NHP models for use in life sciences research. © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society.

Kishi N.,Keio University | Sato K.,Central Institute for Experimental Animals | Sasaki E.,Keio University | Sasaki E.,Central Institute for Experimental Animals | Okano H.,Keio University
Development Growth and Differentiation | Year: 2014

The common marmoset (Callithrix jacchus) is a small New World primate; it originally comes from the Atlantic coastal forests in northeastern Brazil. It has been attracting much attention in the biomedical research field because of its size, availability, and unique biological characteristics. Its endocrinological and behavioral similarity to humans, comparative ease in handling, and high reproductive efficiency are very advantageous for neuroscience research. Recently, we developed transgenic common marmosets with germline transmission, and this technological breakthrough provides a potential paradigm shift by enabling researchers to investigate complex biological phenomena using genetically-modified non-human primates. In this review, we summarize recent progress in marmoset research, and also discuss a potential application of genome editing tools that should be useful toward the generation of knock-out/knock-in marmoset models. © 2014 Japanese Society of Developmental Biologists.

An object of the present invention is to provide a method for introducing a gene into an embryo for production of a human disease model primate animal using a non-human primate animal such as a marmoset. The present invention relates to a method for introducing a foreign gene into an early embryo of a non-human primate animal, which comprises placing early embryos of a non-human primate in a 0.2 M to 0.3 M sucrose solution, so as to increase the volume of the perivitelline spaces, and then injecting a viral vector containing a human foreign gene operably linked to a promoter into the perivitelline spaces of the early embryos.

Ito R.,Central Institute for Experimental Animals | Takahashi T.,Central Institute for Experimental Animals | Katano I.,Central Institute for Experimental Animals | Ito M.,Central Institute for Experimental Animals
Cellular and Molecular Immunology | Year: 2012

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγnull gene (e.g., NOD/SCID/γc null and Rag2null γcnull mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γcnull and Rag2null γcnull mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized. © 2012 CSI and USTC. All rights reserved.

In research into type 2 diabetes, diet-based approaches, i.e., nutritional intake, are important approaches for therapeutic research. We would like to make the following two proposals from the standpoint of laboratory animal science for reproducible animal studies using type 2 diabetes mouse models. These include congenic strains of diabetes mouse models and improvement of diets used in daily care and management. In this research, the Irs2homo-knockout mouse with both impaired glucose tolerance and insulin resistance, and thus type 2 diabetes, was established as a congenic strain. The effect of the genetic background on the onset of diabetes was examined. Next, we discussed which diets are appropriate for general care and management of mouse models in which the pathophysiology is controlled by nutritional conditions. Therefore, we prepared diets by converting the current Japanese and US diets to mice and adjusting the diet contents accordingly. We compared the insulin signals such as those of the liver, pancreas and white fat. We were thus able to establish an evaluation system closer to diabetes in the current population. Using this data as an example, we should consider the quality and ordinary diet of animals as important factors in animal experiments.

Ando K.,Central Institute for Experimental Animals | Inoue T.,Central Institute for Experimental Animals | Itoh T.,Central Institute for Experimental Animals
Pharmacology Biochemistry and Behavior | Year: 2014

l-DOPA is the gold standard for treatment of Parkinson's disease (PD). However, the drug produces some serious side effects, including dyskinesia, which is characterized by repetitive involuntary movements - including chorea. In the present preclinical study using a nonhuman primate model, dyskinesia caused by repeated l-DOPA administration was investigated in the context of behavioral sensitization by objectively quantifying motor activity in the common marmoset of PD model (the Parkinsonian marmoset). Twelve male Parkinsonian marmosets previously treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and six intact male marmosets were used. The motor activity of each marmoset was measured using infrared sensors attached to each individual living cage. Parkinsonian marmosets (n = 6) exhibited behavioral sensitization (enhanced motor activity) in 10 weeks upon oral administration of l-DOPA (10 mg/kg per day on 3 days/week). These animals also exhibited dyskinesia characterized by repetitive rapid movements including chorea in 6-10 weeks. Neither behavioral sensitization nor dyskinesia was observed in Parkinsonian marmosets given vehicle and in intact marmosets given l-DOPA at the same dose (both n = 6 each). Behavioral sensitization was detected sensitively and objectively on motor activity only in Parkinsonian marmosets given repeated l-DOPA at a similar dose used in PD patients. The behavioral feature of the marmosets was dyskinesia similar to that of PD patients but appeared earlier than would be manifested in humans. In spite of statistically significant behavioral sensitization, some marmosets did not exhibit dyskinesia in the present limited l-DOPA administration period. Although both commonalities and differences may exist between behavioral sensitization and dyskinesia, behavioral sensitization is considered to be an objective, quantitative, sensitive and predictive measure of behavioral mechanism underlying dyskinesia in preclinical studies in evaluating compounds. © 2014 Elsevier Inc. All rights reserved.

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