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Marcó S, Muñoz S, Bosch F, Jimenez V. Rat models of musculoskeletal lysosomal storage disorders and their role in pre-clinical evaluation of gene therapy approaches. Mamm Genome 2025:10.1007/s00335-025-10121-3. [PMID: 40100425 DOI: 10.1007/s00335-025-10121-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 03/05/2025] [Indexed: 03/20/2025]
Abstract
Mice have been a cornerstone of biomedical research for decades for studying a wide range of biological processes, disease mechanisms, and the assessment of therapies. Moreover, mice present several practical advantages such as small size, low cost and ease of genetic manipulation. While mice offer numerous benefits, for certain disease areas, rat models provide a closer representation of human disease progression, offering better insights for translational research and therapeutic development. This closer resemblance is particularly important for research focusing on diseases involving the cardiovascular and musculoskeletal system. In rats, the pathophysiology of these diseases mirrors the clinical alterations observed in humans. This review focuses on the key phenotypic differences between mouse and rat models of lysosomal storage disorders that specifically manifest with cardiac, skeletal muscle, and bone and joint involvement (Pompe and Danon diseases, and Maroteaux-Lamy and Morquio A syndromes). Furthermore, we discuss the therapeutic potential of various adeno-associated viral vector-mediated gene therapies that have been evaluated in these rat models, highlighting their contributions to advancing treatment options for these debilitating conditions.
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Affiliation(s)
- Sara Marcó
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Sergio Muñoz
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Fatima Bosch
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Veronica Jimenez
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
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2
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Nicotra R, Lutz C, Messal HA, Jonkers J. Rat Models of Hormone Receptor-Positive Breast Cancer. J Mammary Gland Biol Neoplasia 2024; 29:12. [PMID: 38913216 PMCID: PMC11196369 DOI: 10.1007/s10911-024-09566-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/29/2024] [Accepted: 06/07/2024] [Indexed: 06/25/2024] Open
Abstract
Hormone receptor-positive (HR+) breast cancer (BC) is the most common type of breast cancer among women worldwide, accounting for 70-80% of all invasive cases. Patients with HR+ BC are commonly treated with endocrine therapy, but intrinsic or acquired resistance is a frequent problem, making HR+ BC a focal point of intense research. Despite this, the malignancy still lacks adequate in vitro and in vivo models for the study of its initiation and progression as well as response and resistance to endocrine therapy. No mouse models that fully mimic the human disease are available, however rat mammary tumor models pose a promising alternative to overcome this limitation. Compared to mice, rats are more similar to humans in terms of mammary gland architecture, ductal origin of neoplastic lesions and hormone dependency status. Moreover, rats can develop spontaneous or induced mammary tumors that resemble human HR+ BC. To date, six different types of rat models of HR+ BC have been established. These include the spontaneous, carcinogen-induced, transplantation, hormone-induced, radiation-induced and genetically engineered rat mammary tumor models. Each model has distinct advantages, disadvantages and utility for studying HR+ BC. This review provides a comprehensive overview of all published models to date.
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Affiliation(s)
- Raquel Nicotra
- Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, Netherlands
- Oncode Institute, Amsterdam, Netherlands
| | - Catrin Lutz
- Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, Netherlands.
- Oncode Institute, Amsterdam, Netherlands.
| | - Hendrik A Messal
- Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, Netherlands.
- Oncode Institute, Amsterdam, Netherlands.
| | - Jos Jonkers
- Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, Netherlands.
- Oncode Institute, Amsterdam, Netherlands.
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3
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Ma X, Xing Y, Zhai R, Du Y, Yan H. Development and advancements in rodent MRI-based brain atlases. Heliyon 2024; 10:e27421. [PMID: 38510053 PMCID: PMC10950579 DOI: 10.1016/j.heliyon.2024.e27421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 02/15/2024] [Accepted: 02/28/2024] [Indexed: 03/22/2024] Open
Abstract
Rodents, particularly mice and rats, are extensively utilized in fundamental neuroscience research. Brain atlases have played a pivotal role in this field, evolving from traditional printed histology atlases to digital atlases incorporating diverse imaging datasets. Magnetic resonance imaging (MRI)-based brain atlases, also known as brain maps, have been employed in specific studies. However, the existence of numerous versions of MRI-based brain atlases has impeded their standardized application and widespread use, despite the consensus within the academic community regarding their significance in mice and rats. Furthermore, there is a dearth of comprehensive and systematic reviews on MRI-based brain atlases for rodents. This review aims to bridge this gap by providing a comprehensive overview of the advancements in MRI-based brain atlases for rodents, with a specific focus on mice and rats. It seeks to explore the advantages and disadvantages of histologically printed brain atlases in comparison to MRI brain atlases, delineate the standardized methods for creating MRI brain atlases, and summarize their primary applications in neuroscience research. Additionally, this review aims to assist researchers in selecting appropriate versions of MRI brain atlases for their studies or refining existing MRI brain atlas resources, thereby facilitating the development and widespread adoption of standardized MRI-based brain atlases in rodents.
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Affiliation(s)
- Xiaoyi Ma
- Department of Geriatrics, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080, China
| | - Yao Xing
- School of Information Science and Technology, Fudan University, Shanghai, 200433, China
- Wuhan United Imaging Life Science Instrument Co., Ltd., Wuhan, 430071, China
| | - Renkuan Zhai
- Wuhan United Imaging Life Science Instrument Co., Ltd., Wuhan, 430071, China
| | - Yingying Du
- Wuhan United Imaging Life Science Instrument Co., Ltd., Wuhan, 430071, China
| | - Huanhuan Yan
- Shenzhen United Imaging Research Institute of Innovative Medical Equipment, Shenzhen, 518048, China
- Paul C. Lauterbur Research Center for Biomedical Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China
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4
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Bu W, Li Y. Advances in Immunocompetent Mouse and Rat Models. Cold Spring Harb Perspect Med 2024; 14:a041328. [PMID: 37217281 PMCID: PMC10810718 DOI: 10.1101/cshperspect.a041328] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Rodent models of breast cancer have played critical roles in our understanding of breast cancer development and progression as well as preclinical testing of cancer prevention and therapeutics. In this article, we first review the values and challenges of conventional genetically engineered mouse (GEM) models and newer iterations of these models, especially those with inducible or conditional regulation of oncogenes and tumor suppressors. Then, we discuss nongermline (somatic) GEM models of breast cancer with temporospatial control, made possible by intraductal injection of viral vectors to deliver oncogenes or to manipulate the genome of mammary epithelial cells. Next, we introduce the latest development in precision editing of endogenous genes using in vivo CRISPR-Cas9 technology. We conclude with the recent development in generating somatic rat models for modeling estrogen receptor-positive breast cancer, something that has been difficult to accomplish in mice.
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Affiliation(s)
- Wen Bu
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Yi Li
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA
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Hu S, Li Z, Liu H, Cao W, Meng Y, Liu C, He S, Lin Q, Shang M, Lin F, Yi N, Wang H, Sachinidis A, Ying Q, Li L, Peng L. Kcnh2 deletion is associated with rat embryonic development defects via destruction of KCNH2‑integrin β1 complex. Int J Mol Med 2024; 53:14. [PMID: 38063256 PMCID: PMC10760793 DOI: 10.3892/ijmm.2023.5338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Accepted: 10/06/2023] [Indexed: 12/18/2023] Open
Abstract
The Kv11.1 potassium channel encoded by the Kcnh2 gene is crucial in conducting the rapid delayed rectifier K+ current in cardiomyocytes. Homozygous mutation in Kcnh2 is embryonically lethal in humans and mice. However, the molecular signaling pathway of intrauterine fetal loss is unclear. The present study generated a Kcnh2 knockout rat based on edited rat embryonic stem cells (rESCs). Kcnh2 knockout was embryonic lethal on day 11.5 of development due to a heart configuration defect. Experiments with human embryonic heart single cells (6.5‑7 weeks post‑conception) suggested that potassium voltage‑gated channel subfamily H member 2 (KCNH2) plays a crucial role in the development of compact cardiomyocytes. By contrast, apoptosis was found to be triggered in the homozygous embryos, which could be attributed to the failure of KCNH2 to form a complex with integrin β1 that was essential for preventing the process of apoptosis via inhibition of forkhead box O3A. Destruction of the KCNH2/integrin β1 complex reduced the phosphorylation level of AKT and deactivated the glycogen synthase kinase 3 β (GSK‑3β)/β‑catenin pathway, which caused early developmental abnormalities in rats. The present work reveals a basic mechanism by which KCNH2 maintains intact embryonic heart development.
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Affiliation(s)
- Sangyu Hu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Zhigang Li
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Huan Liu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Wenze Cao
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Yilei Meng
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Chang Liu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Siyu He
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Qin Lin
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Mengyue Shang
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Fang Lin
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Na Yi
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Hanrui Wang
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Agapios Sachinidis
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Center for Physiology, Working Group Sachinidis, Center for Molecular Medicine, D-50931 Cologne, Germany
| | - Qilong Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Li Li
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
- Department of Medical Genetics Tongji University School of Medicine, Shanghai 200331, P.R. China
| | - Luying Peng
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
- Department of Medical Genetics Tongji University School of Medicine, Shanghai 200331, P.R. China
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Men H, Amos-Landgraf JM, Bryda EC, Franklin CL. KSOM-R supports both mouse and rat preimplantation embryo development in vitro. Theriogenology 2023; 198:69-74. [PMID: 36563630 PMCID: PMC9870949 DOI: 10.1016/j.theriogenology.2022.12.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 12/14/2022] [Accepted: 12/15/2022] [Indexed: 12/23/2022]
Abstract
A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2-8 °C for consistency. We first examined the effects of newly made (≤1 month at 2-8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes. We then investigated the effect of extended shelf life (6 months at 2-8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2-8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2-8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2-8 °C) and newly made (≤1 month at 2-8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2-8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2-8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2-8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.
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Affiliation(s)
- Hongsheng Men
- Mutant Mouse Resource and Research Center, USA; Rat Resource and Research Center, USA; Department of Veterinary Pathobiology, University of Missouri-Columbia, 4011 Discovery Drive, Columbia, MO, USA.
| | - James M Amos-Landgraf
- Mutant Mouse Resource and Research Center, USA; Rat Resource and Research Center, USA; Department of Veterinary Pathobiology, University of Missouri-Columbia, 4011 Discovery Drive, Columbia, MO, USA
| | - Elizabeth C Bryda
- Mutant Mouse Resource and Research Center, USA; Rat Resource and Research Center, USA; Department of Veterinary Pathobiology, University of Missouri-Columbia, 4011 Discovery Drive, Columbia, MO, USA
| | - Craig L Franklin
- Mutant Mouse Resource and Research Center, USA; Rat Resource and Research Center, USA; Department of Veterinary Pathobiology, University of Missouri-Columbia, 4011 Discovery Drive, Columbia, MO, USA
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Transition from Animal-Based to Human Induced Pluripotent Stem Cells (iPSCs)-Based Models of Neurodevelopmental Disorders: Opportunities and Challenges. Cells 2023; 12:cells12040538. [PMID: 36831205 PMCID: PMC9954744 DOI: 10.3390/cells12040538] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 01/25/2023] [Accepted: 02/02/2023] [Indexed: 02/11/2023] Open
Abstract
Neurodevelopmental disorders (NDDs) arise from the disruption of highly coordinated mechanisms underlying brain development, which results in impaired sensory, motor and/or cognitive functions. Although rodent models have offered very relevant insights to the field, the translation of findings to clinics, particularly regarding therapeutic approaches for these diseases, remains challenging. Part of the explanation for this failure may be the genetic differences-some targets not being conserved between species-and, most importantly, the differences in regulation of gene expression. This prompts the use of human-derived models to study NDDS. The generation of human induced pluripotent stem cells (hIPSCs) added a new suitable alternative to overcome species limitations, allowing for the study of human neuronal development while maintaining the genetic background of the donor patient. Several hIPSC models of NDDs already proved their worth by mimicking several pathological phenotypes found in humans. In this review, we highlight the utility of hIPSCs to pave new paths for NDD research and development of new therapeutic tools, summarize the challenges and advances of hIPSC-culture and neuronal differentiation protocols and discuss the best way to take advantage of these models, illustrating this with examples of success for some NDDs.
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8
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Goto T, Yogo K, Hochi S, Hirabayashi M. Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease. Mol Biol Rep 2023; 50:1231-1239. [PMID: 36441374 DOI: 10.1007/s11033-022-08054-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Accepted: 10/19/2022] [Indexed: 11/30/2022]
Abstract
BACKGROUND The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. METHODS AND RESULTS We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. CONCLUSIONS The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.
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Affiliation(s)
- Teppei Goto
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan.,Laboratory for Comparative Connectomics, RIKEN Center for Biosystems Dynamics Research, 650-0047, Kobe, Hyogo, Japan
| | - Kyoko Yogo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan
| | - Shinichi Hochi
- Faculty of Textile Science and Technology, Shinshu University, 386-8567, Ueda, Nagano, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan. .,The Graduate University of Advanced Studies, 444-8787, Okazaki, Aichi, Japan.
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Men H, Davis DJ, Bryda EC. Gene Targeting in Rat Embryonic Stem Cells. Methods Mol Biol 2023; 2631:341-353. [PMID: 36995676 DOI: 10.1007/978-1-0716-2990-1_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.
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Affiliation(s)
- Hongsheng Men
- Rat Resource and Research Center, University of Missouri, Columbia, MO, USA.
| | - Daniel J Davis
- Animal Modeling Core, University of Missouri, Columbia, MO, USA
| | - Elizabeth C Bryda
- Rat Resource and Research Center, University of Missouri, Columbia, MO, USA
- Animal Modeling Core, University of Missouri, Columbia, MO, USA
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10
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Bryda EC, Men H, Stone BJ. Rat Embryonic Stem Cell Transgenesis. Methods Mol Biol 2023; 2631:355-370. [PMID: 36995677 DOI: 10.1007/978-1-0716-2990-1_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.
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Affiliation(s)
- Elizabeth C Bryda
- University of Missouri, Rat Resource and Research Center, Columbia, MO, USA.
| | - Hongsheng Men
- University of Missouri, Rat Resource and Research Center, Columbia, MO, USA
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Liu C, Cui Z, Yan Y, Wu NL, Li L, Ying Q, Peng L. An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification. Biochem Biophys Res Commun 2021; 571:8-13. [PMID: 34298338 DOI: 10.1016/j.bbrc.2021.07.053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Accepted: 07/15/2021] [Indexed: 11/25/2022]
Abstract
Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.
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Affiliation(s)
- Chang Liu
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China
| | - Zhonglin Cui
- Division of Hepatobiliopancreatic Surgery, Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Youzhen Yan
- USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Nancy L Wu
- USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Li Li
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China; Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing, 100730, China.
| | - Qilong Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA; USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA.
| | - Luying Peng
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China; Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing, 100730, China.
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12
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Heffner KM, Wang Q, Hizal DB, Can Ö, Betenbaugh MJ. Glycoengineering of Mammalian Expression Systems on a Cellular Level. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2021. [PMID: 29532110 DOI: 10.1007/10_2017_57] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Mammalian expression systems such as Chinese hamster ovary (CHO), mouse myeloma (NS0), and human embryonic kidney (HEK) cells serve a critical role in the biotechnology industry as the production host of choice for recombinant protein therapeutics. Most of the recombinant biologics are glycoproteins that contain complex oligosaccharide or glycan attachments representing a principal component of product quality. Both N-glycans and O-glycans are present in these mammalian cells, but the engineering of N-linked glycosylation is of critical interest in industry and many efforts have been directed to improve this pathway. This is because altering the N-glycan composition can change the product quality of recombinant biotherapeutics in mammalian hosts. In addition, sialylation and fucosylation represent components of the glycosylation pathway that affect circulatory half-life and antibody-dependent cellular cytotoxicity, respectively. In this chapter, we first offer an overview of the glycosylation, sialylation, and fucosylation networks in mammalian cells, specifically CHO cells, which are extensively used in antibody production. Next, genetic engineering technologies used in CHO cells to modulate glycosylation pathways are described. We provide examples of their use in CHO cell engineering approaches to highlight these technologies further. Specifically, we describe efforts to overexpress glycosyltransferases and sialyltransfereases, and efforts to decrease sialidase cleavage and fucosylation. Finally, this chapter covers new strategies and future directions of CHO cell glycoengineering, such as the application of glycoproteomics, glycomics, and the integration of 'omics' approaches to identify, quantify, and characterize the glycosylated proteins in CHO cells. Graphical Abstract.
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Affiliation(s)
- Kelley M Heffner
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Qiong Wang
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Deniz Baycin Hizal
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Özge Can
- Department of Medical Engineering, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey
| | - Michael J Betenbaugh
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
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13
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Soto DA, Navarro M, Zheng C, Halstead MM, Zhou C, Guiltinan C, Wu J, Ross PJ. Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells. Sci Rep 2021; 11:11045. [PMID: 34040070 PMCID: PMC8155104 DOI: 10.1038/s41598-021-90422-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Accepted: 05/05/2021] [Indexed: 02/04/2023] Open
Abstract
Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.
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Affiliation(s)
- Delia Alba Soto
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA
| | - Micaela Navarro
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA
- Instituto de Investigaciones Biotecnológicas 'Dr Rodolfo Ugalde', UNSAM-CONICET, Buenos Aires, Argentina
| | - Canbin Zheng
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA
| | | | - Chuan Zhou
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA
| | - Carly Guiltinan
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA
| | - Pablo Juan Ross
- Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA.
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14
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Hume DA, Caruso M, Keshvari S, Patkar OL, Sehgal A, Bush SJ, Summers KM, Pridans C, Irvine KM. The Mononuclear Phagocyte System of the Rat. THE JOURNAL OF IMMUNOLOGY 2021; 206:2251-2263. [PMID: 33965905 DOI: 10.4049/jimmunol.2100136] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Accepted: 03/01/2021] [Indexed: 12/14/2022]
Abstract
The laboratory rat continues to be the model of choice for many studies of physiology, behavior, and complex human diseases. Cells of the mononuclear phagocyte system (MPS; monocytes, macrophages, and dendritic cells) are abundant residents in every tissue in the body and regulate postnatal development, homeostasis, and innate and acquired immunity. Recruitment and proliferation of MPS cells is an essential component of both initiation and resolution of inflammation. The large majority of current knowledge of MPS biology is derived from studies of inbred mice, but advances in technology and resources have eliminated many of the advantages of the mouse as a model. In this article, we review the tools available and the current state of knowledge of development, homeostasis, regulation, and diversity within the MPS of the rat.
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Affiliation(s)
- David A Hume
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Melanie Caruso
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Sahar Keshvari
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Omkar L Patkar
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Anuj Sehgal
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Stephen J Bush
- Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
| | - Kim M Summers
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
| | - Clare Pridans
- Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom.,Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Katharine M Irvine
- Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia
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15
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Lee JM, Kim U, Yang H, Ryu B, Kim J, Sakuma T, Yamamoto T, Park JH. TALEN-mediated generation of Nkx3.1 knockout rat model. Prostate 2021; 81:182-193. [PMID: 33368416 DOI: 10.1002/pros.24095] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Revised: 11/16/2020] [Accepted: 11/21/2020] [Indexed: 11/05/2022]
Abstract
BACKGROUND Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans. METHODS Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology. RESULTS Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors. CONCLUSIONS We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.
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Affiliation(s)
- Ji Min Lee
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Ukjin Kim
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Hyokyung Yang
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Bokyeong Ryu
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Jin Kim
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
| | - Tetsushi Sakuma
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
| | - Takashi Yamamoto
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
| | - Jae-Hak Park
- Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea
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16
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Lanz B, Abaei A, Braissant O, Choi IY, Cudalbu C, Henry PG, Gruetter R, Kara F, Kantarci K, Lee P, Lutz NW, Marjańska M, Mlynárik V, Rasche V, Xin L, Valette J. Magnetic resonance spectroscopy in the rodent brain: Experts' consensus recommendations. NMR IN BIOMEDICINE 2020; 34:e4325. [PMID: 33565219 PMCID: PMC9429976 DOI: 10.1002/nbm.4325] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Revised: 03/29/2020] [Accepted: 04/30/2020] [Indexed: 05/21/2023]
Abstract
In vivo MRS is a non-invasive measurement technique used not only in humans, but also in animal models using high-field magnets. MRS enables the measurement of metabolite concentrations as well as metabolic rates and their modifications in healthy animals and disease models. Such data open the way to a deeper understanding of the underlying biochemistry, related disturbances and mechanisms taking place during or prior to symptoms and tissue changes. In this work, we focus on the main preclinical 1H, 31P and 13C MRS approaches to study brain metabolism in rodent models, with the aim of providing general experts' consensus recommendations (animal models, anesthesia, data acquisition protocols). An overview of the main practical differences in preclinical compared with clinical MRS studies is presented, as well as the additional biochemical information that can be obtained in animal models in terms of metabolite concentrations and metabolic flux measurements. The properties of high-field preclinical MRS and the technical limitations are also described.
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Affiliation(s)
- Bernard Lanz
- Laboratory for Functional and Metabolic Imaging (LIFMET), Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
- Sir Peter Mansfield Imaging Centre, School of Medicine, University of Nottingham, Nottingham, United Kingdom
| | - Alireza Abaei
- Core Facility Small Animal Imaging, Ulm University, Ulm, Germany
| | - Olivier Braissant
- Service of Clinical Chemistry, University of Lausanne and University Hospital of Lausanne, Lausanne, Switzerland
| | - In-Young Choi
- Department of Neurology, University of Kansas Medical Center, Kansas City, Kansas, US
| | - Cristina Cudalbu
- Centre d'Imagerie Biomedicale (CIBM), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
| | - Pierre-Gilles Henry
- Center for Magnetic Resonance Research, Department of Radiology, University of Minnesota, Minneapolis, Minnesota, US
| | - Rolf Gruetter
- Laboratory for Functional and Metabolic Imaging (LIFMET), Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Firat Kara
- Department of Radiology, Mayo Clinic, Rochester, Minnesota, US
| | - Kejal Kantarci
- Department of Radiology, Mayo Clinic, Rochester, Minnesota, US
| | - Phil Lee
- Department of Radiology, University of Kansas Medical Center, Kansas City, Kansas, US
| | - Norbert W Lutz
- CNRS, CRMBM, Aix-Marseille University, Marseille, France
| | - Małgorzata Marjańska
- Center for Magnetic Resonance Research, Department of Radiology, University of Minnesota, Minneapolis, Minnesota, US
| | - Vladimír Mlynárik
- High Field MR Centre, Department of Biomedical Imaging and Image-Guided Therapy, Medical University of Vienna, Vienna, Austria
| | - Volker Rasche
- Core Facility Small Animal Imaging, Ulm University, Ulm, Germany
| | - Lijing Xin
- Centre d'Imagerie Biomedicale (CIBM), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
| | - Julien Valette
- Commissariat à l'Energie Atomique et aux Energies Alternatives, MIRCen, Fontenay-aux-Roses, France
- Neurodegenerative Diseases Laboratory, Centre National de la Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, UMR 9199, Fontenay-aux-Roses, France
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17
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Park JS, Kim JI, Lim HJ, Ryu SK, Kwon E, Han KM, Nam KT, Lee HW, Kang BC. Differential manifestation of ocular phenotypes in TALEN-mediated p19 arf knockout FVB/N and C57BL/6J mouse lines. Genes Genomics 2020; 42:1023-1033. [PMID: 32712838 DOI: 10.1007/s13258-020-00959-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Accepted: 06/09/2020] [Indexed: 11/29/2022]
Abstract
BACKGROUND p19arf, primarily known as a tumor suppressor, has also been reported to play an essential role in normal development of mouse eyes. Consistently, lack of p19arf has been associated with ocular defects, but the mixed background of the knockout (KO) mouse strain used raised a concern on the accuracy of the phenotypes observed in association with the targeted gene due to genetic heterogeneity. OBJECT We carried out a study to investigate into the effect of genetic background on the manifestation of p19arf KO associated phenotypes. METHODS We characterized the phenotypes of novel p19arf KO mouse lines generated in FVB/N and C57BL/6J using a transcription activator-like effector nuclease (TALEN) system in comparison to the reported phenotypes of three other p19arf-deficient mouse lines generated using homologous recombination. RESULTS Ninety-five percent of FVB/N-p19arf KO mice showed ocular opacity from week 4 after birth which worsened rapidly until week 6, while such abnormality was absent in C57BL/6J-p19arf KO mice up to the age of 26 weeks. Histopathological analysis revealed retrolental masses and dysplasia in the retinal layer in FVB/N-p19arf KO mice from week 4. Besides these, both strains developed normally from birth to week 26 without increased tumorigenesis except for a subcutaneous tumor found in a C57BL/6J-p19arf KO mouse. CONCLUSION Our findings demonstrated surprisingly variable manifestation of p19arf-linked phenotypes between FVB/N and C57BL/6J mice, and furthermore between our mouse lines and the established lines, indicating a critical impact of genetic background on functional study of genes using gene targeting strategies in mice.
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Affiliation(s)
- Jin-Sung Park
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Joo-Il Kim
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.,Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Hyun-Jin Lim
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.,Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Soo-Kyung Ryu
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Euna Kwon
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Kang-Min Han
- Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.,Department of Pathology, Dongguk University Ilsan Hospital, Goyang, Republic of Korea
| | - Ki-Taek Nam
- College of Medicine Severance Biomedical Science Institute, Yonsei University, Seoul, Republic of Korea
| | - Han-Woong Lee
- Department of Biochemistry, Yonsei University, Seoul, Republic of Korea
| | - Byeong-Cheol Kang
- Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea. .,Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea. .,Biomedical Center for Animal Resource and Development, Seoul National University College of Medicine, Seoul, Republic of Korea. .,Designed Animal Resource Center, Institute of GreenBio Science and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea.
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18
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Wang D, Wang Y, Liu H, Tong C, Ying Q, Sachinidis A, Li L, Peng L. Laminin promotes differentiation of rat embryonic stem cells into cardiomyocytes by activating the integrin/FAK/PI3K p85 pathway. J Cell Mol Med 2019; 23:3629-3640. [PMID: 30907509 PMCID: PMC6484303 DOI: 10.1111/jcmm.14264] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Revised: 01/02/2019] [Accepted: 01/10/2019] [Indexed: 12/27/2022] Open
Abstract
The generation of germline competent rat embryonic stem cells (rESCs) allows the study of their lineage commitment. Here, we developed a highly efficient system for rESC-derived cardiomyocytes, and even the formation of three-dimensional (3D)-like cell clusters with cTNT and α-Actinin. We have validated that laminin can interact with membrane integrin to promote the phosphorylation of both phosphatidylinositol 3-kinase (PI3K) p85 and the focal adhesion kinase (FAK). In parallel, GATA4 was up-regulated. Upon inhibiting the integrin, laminin loses the effect on cardiomyocyte differentiation, accompanied with a down-regulation of phosphorylation level of PI3K p85 and FAK. Meanwhile, the expression of Gata4 was inhibited as well. Taken together, laminin is a crucial component in the differentiation of rESCs into cardiomyocytes through increasing their proliferation via interacting with integrin pathway. These results provide new insights into the pathways mediated by extracellular laminin involved in the fate of rESC-derived cardiomyocytes.
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Affiliation(s)
- Duo Wang
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Department of Pathology and PathophysiologyTongji University School of MedicineShanghaiChina
| | - Yumei Wang
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Department of Pathology and PathophysiologyTongji University School of MedicineShanghaiChina
| | - Huan Liu
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Department of Pathology and PathophysiologyTongji University School of MedicineShanghaiChina
| | - Chang Tong
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
| | - Qilong Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCalifornia
| | - Agapios Sachinidis
- Institute of Neurophysiology and Center for Molecular MedicineUniversity of CologneCologneGermany
| | - Li Li
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Department of Pathology and PathophysiologyTongji University School of MedicineShanghaiChina
| | - Luying Peng
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Research Center for Translational Medicine, Shanghai East HospitalTongji University School of MedicineShanghaiChina
- Department of Pathology and PathophysiologyTongji University School of MedicineShanghaiChina
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19
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Phenotyping analysis of p53 knockout mice produced by gene editing and comparison with conventional p53 knockout mice. Genes Genomics 2019; 41:701-712. [PMID: 30989490 DOI: 10.1007/s13258-019-00785-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2018] [Accepted: 01/03/2019] [Indexed: 02/07/2023]
Abstract
BACKGROUND Knockout (KO) mice developed by homologous recombination (HR) have become useful tools to elucidate gene function. However, HR has low KO efficiency and is time-consuming, labor-intensive, and expensive. 'Gene editing' has received much attention for efficient genetic manipulation. OBJECTIVE As generation of KO mice is simplified, KO mice produced by HR can be feasibly reproduced using gene editing. However, phenotyping analysis and comparison between KO mice produced by these two techniques is necessary. METHODS We generated p53 KO mice through gene editing and compared their phenotype with the already reported HR-mediated p53 KO mice. RESULTS Tumors occurred in 36 (73%) of 49 homozygous KO mice and the mean age of occurrence was 23 weeks, with lymphoma (64%) and sarcoma (23%) being the most common. Tumors were also developed in 12 heterozygous mice and the mean age of occurrence was 40 weeks, with sarcoma (54%) and lymphoma (46%) in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities were found in females compared to in males (P < 0.05, P < 0.001). CONCLUSION We analyzed the basic phenotype of p53 KO mice and observed no significant difference from the conventional HR-mediated p53 KO mice.
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20
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Abstract
In this chapter, we briefly review the use of rats as a genetic model for the study of behavior. Rats were the first mammalian species used for genetic and biological research. Since the development of the first inbred rat strain in 1909, more than 700 unique inbred and outbred rat lines have been generated. Although rats have been somewhat eclipsed by mice in the last few decades, a renewed appreciation of the advantages of rats for behavioral and other types of research is upon us. We briefly review the pertinent characteristics of the rat and highlight the key advantages of using the rat to examine behavioral phenotypes.
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Affiliation(s)
- Yangsu Ren
- Department of Psychiatry, University of California San Diego, La Jolla, CA, USA
| | - Abraham A Palmer
- Department of Psychiatry, University of California San Diego, La Jolla, CA, USA. .,Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.
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21
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Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib. Sci Rep 2018; 8:16150. [PMID: 30385780 PMCID: PMC6212413 DOI: 10.1038/s41598-018-34399-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Accepted: 10/09/2018] [Indexed: 01/20/2023] Open
Abstract
Species generalization in the profound, modality-specific effects of Hedgehog pathway inhibition (HPI) in taste organ homeostasis and sensation is shown. With the HPI, cancer drug sonidegib, we demonstrate that the rat taste system, in addition to mouse, is regulated by Hedgehog signaling. After sonidegib treatment for 16–36 days in rat, there is loss of taste buds (TB) in soft palate, in fungiform (FP) and circumvallate papillae (CV), and elimination of taste responses from chorda tympani and glossopharyngeal nerves. The retained innervation in FP and CV during HPI cannot sustain TB. Responses to tactile stimuli are not altered, and temperature responses are reduced only after 28 days treatment, demonstrating modality-specific effects. Rat FP and neural effects are similar to those in mouse whereas TB and neural response effects from the rat CV are much more severe. When recovery is introduced in mouse after prolonged, 48 days HPI, the TB in CV are restored whereas those in FP are not. Overall, Hedgehog signaling regulation is shown to generalize to the rat taste system, and the modality-specific controls in taste organ sensation are affirmed. The reported, debilitating taste disturbances in patients who use HPI drugs can be better understood based on these data.
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Abstract
Rodents (especially Mus musculus and Rattus norvegicus) have been the most widely used models in biomedical research for many years. A notable shift has taken place over the last two decades, with mice taking a more and more prominent role in biomedical science compared to rats. This shift was primarily instigated by the availability of a much larger genetic toolbox for mice, particularly embryonic-stem-cell-based targeting technology for gene disruption. With the recent emergence of tools for altering the rat genome, notably genome-editing technologies, the technological gap between the two organisms is closing, and it is becoming more important to consider the physiological, anatomical, biochemical and pharmacological differences between rats and mice when choosing the right model system for a specific biological question. The aim of this short review and accompanying poster is to highlight some of the most important differences, and to discuss their impact on studies of human diseases, with a special focus on neuropsychiatric disorders.
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Affiliation(s)
- Bart Ellenbroek
- School of Psychology, Victoria University of Wellington, PO Box 600, Wellington 6041, New Zealand
| | - Jiun Youn
- School of Psychology, Victoria University of Wellington, PO Box 600, Wellington 6041, New Zealand
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23
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Thomas AM, Schwartz MD, Saxe MD, Kilduff TS. Cntnap2 Knockout Rats and Mice Exhibit Epileptiform Activity and Abnormal Sleep-Wake Physiology. Sleep 2017; 40:2661545. [PMID: 28364455 DOI: 10.1093/sleep/zsw026] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/02/2016] [Indexed: 11/12/2022] Open
Abstract
Study Objectives Although recent innovations have enabled modification of the rat genome, it is unclear whether enhanced utility of rodents as human disease models will result. We compared electroencephalogram (EEG) and behavioral phenotypes of rats and mice with homozygous deletion of Cntnap2, a gene associated with cortical dysplasia-focal epilepsy (CDFE) and autism spectrum disorders (ASD). Methods Male contactin-associated protein-like 2 (Cntnap2) knockout (KO) and wild-type (WT) rats and male Cntnap2 KO and WT mice were implanted with telemeters to record EEG, electromyogram, body temperature, and locomotor activity. Animals were subjected to a test battery for ASD-related behaviors, followed by 24-hr EEG recordings that were analyzed for sleep-wake parameters and subjected to spectral analysis. Results Cntnap2 KO rats exhibited severe motor seizures, hyperactivity, and increased consolidation of wakefulness and REM sleep. By contrast, Cntnap2 KO mice demonstrated absence seizure-like events, hypoactivity, and wake fragmentation. Although seizures observed in Cntnap2 KO rats were more similar to those in CDFE patients than in KO mice, neither model fully recapitulated the full spectrum of disease symptoms. However, KOs in both species had reduced spectral power in the alpha (9-12 Hz) range during wake, suggesting a conserved EEG biomarker. Conclusions Deletion of Cntnap2 impacts similar behaviors and EEG measures in rats and mice, but with profound differences in nature and phenotypic severity. These observations highlight the importance of cross-species comparisons to understand conserved gene functions and the limitations of single- species models to provide translational insights relevant to human diseases.
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Affiliation(s)
- Alexia M Thomas
- Biosciences Division, Center for Neuroscience, SRI International, Menlo Park, CA
| | - Michael D Schwartz
- Biosciences Division, Center for Neuroscience, SRI International, Menlo Park, CA
| | - Michael D Saxe
- Pharma Research and Early Development, Neuroscience, Ophthalmology and Rare Disease DTA, F. Hoffmann-La Roche Ltd, Basel, Switzerland
| | - Thomas S Kilduff
- Biosciences Division, Center for Neuroscience, SRI International, Menlo Park, CA
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24
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Endo H, Eto T, Yoshii F, Owada S, Watanabe T, Tatemichi M, Kimura M. The intrauterine environment affects learning ability of Tokai high avoider rat offspring derived using cryopreservation and embryo transfer-mediated reproduction. Biochem Biophys Res Commun 2017; 489:211-216. [PMID: 28552533 DOI: 10.1016/j.bbrc.2017.05.133] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2017] [Accepted: 05/23/2017] [Indexed: 11/16/2022]
Abstract
Embryo transfer (ET) to recipient female animals is a useful technique in biological and experimental animal studies. While cryopreservation of two-cell stage rat embryos and ET to recipient rats are currently well-defined, it is unknown whether these artificial reproductive techniques and maternal factors affect offspring phenotype, particularly higher brain functions. Therefore, we assessed the effects of cryopreservation, ET, and maternal care on learning behaviour of the offspring, using Tokai high avoider (THA) rats that have a high learning ability phenotype. We found that the high learning ability of THA rat offspring was not replicated following ET to surrogate Wistar rats with a low-avoidance phenotype. Additionally, the characteristic phenotype of offspring obtained through mating of ET-derived rats was similar to that of THA rats. A postnatal cross-fostering investigation with the offspring of Wistar and THA rats showed that maternal behaviour, including postnatal care and lactation traits, did not differ between the dams of low-avoidance Wistar rats and THA rats; therefore, learning behaviour was retained in both Wistar and THA rat offspring. We conclude that the offspring phenotype, although unchanged, has an imperceptible effect on the learning ability of ET-derived THA rats through the intrauterine environment of the recipient.
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Affiliation(s)
- Hitoshi Endo
- Center for Molecular Prevention and Environmental Medicine, Department of Preventive Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
| | - Tomoo Eto
- Central Institute for Experimental Animals, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, Japan.
| | - Fumihito Yoshii
- Department of Neurology, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
| | - Satoshi Owada
- Center for Molecular Prevention and Environmental Medicine, Department of Preventive Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
| | - Tetsu Watanabe
- Center for Molecular Prevention and Environmental Medicine, Department of Preventive Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
| | - Masayuki Tatemichi
- Center for Molecular Prevention and Environmental Medicine, Department of Preventive Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
| | - Minoru Kimura
- Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan.
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25
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Wang D, Sang H, Zhang K, Nie Y, Zhao S, Zhang Y, He N, Wang Y, Xu Y, Xie X, Li Z, Liu N. Stat3 phosphorylation is required for embryonic stem cells ground state maintenance in 2i culture media. Oncotarget 2017; 8:31227-31237. [PMID: 28415722 PMCID: PMC5458203 DOI: 10.18632/oncotarget.16112] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2016] [Accepted: 03/01/2017] [Indexed: 12/27/2022] Open
Abstract
Embryonic stem cells (ES cells) can be maintained its undifferentiated state with feeder cells or LIF, which can activate Jak/Stat3 pathway. Recently, it has been reported a new culture condition comprising serum-free medium with ERK and GSK3β inhibitors (2i) could drive ES cells into a state of pluripotency more like inner cell mass (ICM) in mouse blastocysts called ground state. However, although 2i could sustain ES cells self-renewal, LIF is routinely added. The roles of Stat3 activation are still unclear now. Here we investigated whether Jak/Stat3 might also contribute to the induction of ground state pluripotency. We introduced a lentiviral construct with 7-repeat Stat3-binding sequence to drive Renilla luciferase into ES cells, which can be used as a reporter to detect Stat3 activation by noninvasive bioluminescence imaging. Using this ES cells, we investigated the role of Stat3 activation in ground state maintenance. The results showed that Stat3 could be activated by 2i. Stattic, a chemical inhibitor of Stat3 phosphorylation, could effectively inhibit Stat3 activation in ES cells. When Stat3 activation was suppressed, ground state related genes were down regulated, and ES cells could not be maintained the ground state pluripotency even in 2i medium. All of these results indicate Stat3 activation is required in ground state maintenance.
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Affiliation(s)
- Dan Wang
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.,College of Life Sciences, Nankai University, Tianjin, China
| | - Hui Sang
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.,College of Life Sciences, Nankai University, Tianjin, China
| | - Kaiyue Zhang
- School of Medicine, Nankai University, Tianjin, China
| | - Yan Nie
- School of Medicine, Nankai University, Tianjin, China
| | - Shuang Zhao
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.,College of Life Sciences, Nankai University, Tianjin, China
| | - Yan Zhang
- School of Medicine, Nankai University, Tianjin, China.,College of Life Sciences, Nankai University, Tianjin, China
| | - Ningning He
- School of Medicine, Nankai University, Tianjin, China
| | - Yuebing Wang
- School of Medicine, Nankai University, Tianjin, China
| | - Yang Xu
- School of Medicine, Nankai University, Tianjin, China
| | - Xiaoyan Xie
- Stem Cells and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, China
| | - Zongjin Li
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.,School of Medicine, Nankai University, Tianjin, China
| | - Na Liu
- Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.,School of Medicine, Nankai University, Tianjin, China
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26
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May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells. Cells 2017; 6:cells6010005. [PMID: 28178187 PMCID: PMC5371870 DOI: 10.3390/cells6010005] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2016] [Revised: 01/20/2017] [Accepted: 01/30/2017] [Indexed: 12/16/2022] Open
Abstract
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.
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27
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He L, Xu JM, Li H, Zhong F, Liu Z, Li CQ, Dai RP. Moderate hypothermia increased the incidence of delayed paralysis through activation of the spinal microglia in an aortic cross-clamping rat model. Int J Cardiol 2016; 220:454-61. [PMID: 27390969 DOI: 10.1016/j.ijcard.2016.06.169] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 06/24/2016] [Indexed: 10/21/2022]
Abstract
BACKGROUND Hypothermia reduces immediate paralysis during surgical repair of aortic aneurysms. However, it is unknown what the impact of hypothermia is on delayed paralysis, a serious complication of this type of surgery. METHODS Sprague-Dawley rats were subjected to occlusion of the descending aorta at different duration under normothermia (38.0±0.5) or hypothermia (33.0±0.5°). Neurologic function was assessed. Motor neuron number, glial activation, and cytokine expression in the spinal cord were examined. Minocycline was administered perioperatively by intraperitoneal injection in the rats subjected to the aorta occlusion. RESULTS In contrast to normothermia conditions at which immediate paralysis occurred when the duration of aorta occlusion exceeded 11.5min, hypothermia did not induce immediate paralysis if the duration of aorta occlusion was less than 41min. However, delayed paralysis was developed when the duration of aorta occlusion exceeded 18min, and reached peak level when the duration of aorta occlusion was 40min at hypothermia condition. The number of motoneurons was significantly decreased (P<0.05) at 30h postoperation. In addition, microglia was activated, and interleukin-1β and interleukin-6 levels were upregulated, both of which were co-localized in microglia at 24h postoperation in the hypothermia group. Minocycline treatment attenuated the incidence and degree of paralysis but did not decrease the mortality. CONCLUSIONS Hypothermia, a neuroprotective strategy in cardiothoracic surgery, increased the incidence of delayed paralysis through activation of spinal microglia and cytokines. Blocking the activated microglia may be a potential intervention to prevent the incidence of delayed paralysis.
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Affiliation(s)
- Liang He
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China.; Department of Anesthesiology, The Affiliated Hospital of Guilin Medical University, Lequn Road 15#, Guilin 54100, China
| | - Jun-Mei Xu
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China
| | - Hui Li
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China
| | - Feng Zhong
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China
| | - Zhi Liu
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China
| | - Chang-Qi Li
- Department of Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, Changsha 410000, China
| | - Ru-Ping Dai
- Department of Anesthesia, The Second Xiangya Hospital of Central South University, Ren-Min Road 139#, Changsha 410011, China..
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28
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Lan H, Li S, Guo Z, Men H, Wu Y, Li N, Bryda EC, Capecchi MR, Wu S. Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells. FEBS Lett 2016; 590:3416-3424. [PMID: 27597178 PMCID: PMC5129459 DOI: 10.1002/1873-3468.12388] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2016] [Revised: 08/24/2016] [Accepted: 08/25/2016] [Indexed: 11/25/2022]
Abstract
Embryonic stem cell (ES cell)‐based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague–Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self‐excising neomycin (neo)‐positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
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Affiliation(s)
- He Lan
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Shuping Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Zihang Guo
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Hongsheng Men
- Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA
| | - Yuanyuan Wu
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA
| | - Ning Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Elizabeth C Bryda
- Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA
| | - Mario R Capecchi
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA
| | - Sen Wu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
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29
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Li S, Lan H, Men H, Wu Y, Li N, Capecchi MR, Bryda EC, Wu S. Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells. Stem Cells Transl Med 2016; 6:340-351. [PMID: 28191784 PMCID: PMC5442795 DOI: 10.5966/sctm.2015-0390] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2015] [Accepted: 07/28/2016] [Indexed: 01/08/2023] Open
Abstract
Although a variety of reprogramming strategies have been reported to create transgene‐free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene‐free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene‐free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline‐competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague‐Dawley rat embryonic fibroblasts. The obtained transgene‐free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground‐state riPS cells. Stem Cells Translational Medicine2017;6:340–351
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Affiliation(s)
- Shuping Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - He Lan
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Hongsheng Men
- Rat Resource and Research Center, Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - Yuanyuan Wu
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA
| | - Ning Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
| | - Mario R. Capecchi
- Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA
| | - Elizabeth C. Bryda
- Rat Resource and Research Center, Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA
| | - Sen Wu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China
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30
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Periwal V. A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases. Brief Bioinform 2016; 18:698-711. [DOI: 10.1093/bib/bbw052] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Indexed: 12/26/2022] Open
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31
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Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene. SCIENCE CHINA-LIFE SCIENCES 2016; 60:152-157. [DOI: 10.1007/s11427-016-5049-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/12/2015] [Accepted: 11/06/2015] [Indexed: 12/20/2022]
Abstract
Abstract
The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.
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32
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Zhao ZA, Yu Y, Ma HX, Wang XX, Lu X, Zhai Y, Zhang X, Wang H, Li L. The roles of ERAS during cell lineage specification of mouse early embryonic development. Open Biol 2016; 5:rsob.150092. [PMID: 26269429 PMCID: PMC4554925 DOI: 10.1098/rsob.150092] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022] Open
Abstract
Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.
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Affiliation(s)
- Zhen-Ao Zhao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China Institute for Cardiovascular Science and Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University, Suzhou 215000, People's Republic of China
| | - Yang Yu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China Institute of Zoology, University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
| | - Huai-Xiao Ma
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China Institute of Zoology, University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
| | - Xiao-Xiao Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China Institute of Zoology, University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
| | - Xukun Lu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China Institute of Zoology, University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
| | - Yanhua Zhai
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
| | - Xiaoxin Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
| | - Haibin Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
| | - Lei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
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Pradhan BS, Majumdar SS. An Efficient Method for Generation of Transgenic Rats Avoiding Embryo Manipulation. MOLECULAR THERAPY. NUCLEIC ACIDS 2016; 5:e293. [PMID: 27111419 PMCID: PMC5014465 DOI: 10.1038/mtna.2016.9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/11/2015] [Accepted: 01/07/2016] [Indexed: 12/20/2022]
Abstract
Although rats are preferred over mice as an animal model, transgenic animals are generated predominantly using mouse embryos. There are limitations in the generation of transgenic rat by embryo manipulation. Unlike mouse embryos, most of the rat embryos do not survive after male pronuclear DNA injection which reduces the efficiency of generation of transgenic rat by this method. More importantly, this method requires hundreds of eggs collected by killing several females for insertion of transgene to generate transgenic rat. To this end, we developed a noninvasive and deathless technique for generation of transgenic rats by integrating transgene into the genome of the spermatogonial cells by testicular injection of DNA followed by electroporation. After standardization of this technique using EGFP as a transgene, a transgenic disease model displaying alpha thalassemia was successfully generated using rats. This efficient method will ease the generation of transgenic rats without killing the lives of rats while simultaneously reducing the number of rats used for generation of transgenic animal.
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Affiliation(s)
- Bhola Shankar Pradhan
- Cellular Endocrinology Laboratory, National Institute of Immunology, New Delhi, India
| | - Subeer S Majumdar
- Cellular Endocrinology Laboratory, National Institute of Immunology, New Delhi, India
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Liu Z, Brown A, Fisher D, Wu Y, Warren J, Cui X. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons. PLoS One 2016; 11:e0149379. [PMID: 26886559 PMCID: PMC4757100 DOI: 10.1371/journal.pone.0149379] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2015] [Accepted: 02/01/2016] [Indexed: 12/20/2022] Open
Abstract
The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease.
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Affiliation(s)
- Zhenyi Liu
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
| | - Andrew Brown
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
| | - Dan Fisher
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
| | - Yumei Wu
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
| | - Joe Warren
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
| | - Xiaoxia Cui
- SAGE Labs, A Horizon Discovery Group Company, Saint Louis, MO, United States of America
- * E-mail:
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Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning. Neural Plast 2015; 2016:1752176. [PMID: 26843989 PMCID: PMC4710954 DOI: 10.1155/2016/1752176] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Accepted: 10/12/2015] [Indexed: 12/17/2022] Open
Abstract
An increasing body of evidence suggests that mechanisms related to the introduction and repair of DNA double strand breaks (DSBs) may be associated with long-term memory (LTM) processes. Previous studies from our group suggested that factors known to function in DNA recombination/repair machineries, such as DNA ligases, polymerases, and DNA endonucleases, play a role in LTM. Here we report data using C57BL/6 mice showing that the V(D)J recombination-activating gene 1 (RAG1), which encodes a factor that introduces DSBs in immunoglobulin and T-cell receptor genes, is induced in the amygdala, but not in the hippocampus, after context fear conditioning. Amygdalar induction of RAG1 mRNA, measured by real-time PCR, was not observed in context-only or shock-only controls, suggesting that the context fear conditioning response is related to associative learning processes. Furthermore, double immunofluorescence studies demonstrated the neuronal localization of RAG1 protein in amygdalar sections prepared after perfusion and fixation. In functional studies, intra-amygdalar injections of RAG1 gapmer antisense oligonucleotides, given 1 h prior to conditioning, resulted in amygdalar knockdown of RAG1 mRNA and a significant impairment in LTM, tested 24 h after training. Overall, these findings suggest that the V(D)J recombination-activating gene 1, RAG1, may play a role in LTM consolidation.
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The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies. Prog Retin Eye Res 2015; 48:137-59. [DOI: 10.1016/j.preteyeres.2015.04.004] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Revised: 04/12/2015] [Accepted: 04/16/2015] [Indexed: 01/19/2023]
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Kawaharada K, Kawamata M, Ochiya T. Rat embryonic stem cells create new era in development of genetically manipulated rat models. World J Stem Cells 2015; 7:1054-1063. [PMID: 26328021 PMCID: PMC4550629 DOI: 10.4252/wjsc.v7.i7.1054] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2014] [Revised: 01/15/2015] [Accepted: 07/17/2015] [Indexed: 02/07/2023] Open
Abstract
Embryonic stem (ES) cells are isolated from the inner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer gene-modified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.
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Flister MJ, Prokop JW, Lazar J, Shimoyama M, Dwinell M, Geurts A. 2015 Guidelines for Establishing Genetically Modified Rat Models for Cardiovascular Research. J Cardiovasc Transl Res 2015; 8:269-77. [PMID: 25920443 PMCID: PMC4475456 DOI: 10.1007/s12265-015-9626-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 04/15/2015] [Indexed: 12/24/2022]
Abstract
The rat has long been a key physiological model for cardiovascular research, most of the inbred strains having been previously selected for susceptibility or resistance to various cardiovascular diseases (CVD). These CVD rat models offer a physiologically relevant background on which candidates of human CVD can be tested in a more clinically translatable experimental setting. However, a diverse toolbox for genetically modifying the rat genome to test molecular mechanisms has only recently become available. Here, we provide a high-level description of several strategies for developing genetically modified rat models of CVD.
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Affiliation(s)
- Michael J Flister
- Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, WI, USA,
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An alternative pluripotent state confers interspecies chimaeric competency. Nature 2015; 521:316-21. [PMID: 25945737 PMCID: PMC5278765 DOI: 10.1038/nature14413] [Citation(s) in RCA: 194] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2014] [Accepted: 03/18/2015] [Indexed: 12/31/2022]
Abstract
Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.
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Chapman KM, Medrano GA, Jaichander P, Chaudhary J, Waits AE, Nobrega MA, Hotaling JM, Ober C, Hamra FK. Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells. Cell Rep 2015; 10:1828-35. [PMID: 25772367 DOI: 10.1016/j.celrep.2015.02.040] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Revised: 01/12/2015] [Accepted: 02/16/2015] [Indexed: 01/15/2023] Open
Abstract
Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.
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Affiliation(s)
- Karen M Chapman
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Gerardo A Medrano
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Priscilla Jaichander
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jaideep Chaudhary
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Alexandra E Waits
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Marcelo A Nobrega
- Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA
| | - James M Hotaling
- Department of Surgery (Urology), University of Utah School of Medicine, Salt Lake City, UT 84134, USA
| | - Carole Ober
- Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA
| | - F Kent Hamra
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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Huang G, Yan H, Ye S, Tong C, Ying QL. STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates. Stem Cells 2014; 32:1149-60. [PMID: 24302476 DOI: 10.1002/stem.1609] [Citation(s) in RCA: 116] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2013] [Revised: 10/16/2013] [Accepted: 10/30/2013] [Indexed: 12/22/2022]
Abstract
STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule-based system to post-translationally modulate the quantity of transgenic STAT3 in STAT3(-/-) mESCs. We demonstrated that pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naïve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate.
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Affiliation(s)
- Guanyi Huang
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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42
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Pan A, Weintraub NL, Tang Y. Enhancing stem cell survival in an ischemic heart by CRISPR-dCas9-based gene regulation. Med Hypotheses 2014; 83:702-5. [PMID: 25459138 DOI: 10.1016/j.mehy.2014.09.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2014] [Accepted: 09/29/2014] [Indexed: 12/18/2022]
Abstract
Ischemic heart disease has remained the number one killer around the world for over the past 20 years. While stem cell therapy has become a promising new frontier to repair the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Strategies to genetically modify stem cells to activate pro-survival and anti-apoptotic and anti-inflammatory pathways, such as Akt and heme oxygenase-1, have been shown to improve the lifespan of transplanted stem cells within the ischemic myocardium, but constitutive overexpression of these pathways at high levels has been shown to have side effects. Therefore, more specific and controlled gene activation would be necessary. Current techniques used for gene regulation include zinc finger and TALE proteins, but there are still disadvantages to each of these methods, such as ease and cost of use. Also, those methods use synthesized promoters to express synthesized cDNA, which lack regulatory elements, including introns and 3' untranslated regions for microRNA mediated post-transcriptional regulation. A new novel technique, the CRISPR/dCas9 system, was recently developed as a simple and efficient method for endogenous gene regulation. With its use of single guide chimeric RNA's (sgRNA's), this system has been shown to provide a high level of specificity and efficiency. When targeting different loci, past studies have found that the CRISPR/dCas9 system can activate gene expression at varying levels. In addition, this system makes use of the genome's endogenous regulatory elements, such as the aforementioned introns and 3' UTR's, which can help provide a safer method of gene activation. If targeted to a gene promoting cellular survival or decreasing cell death, it could potentially improve stem cell longevity in a more efficient and controllable manner. As a result, our hypothesis is to use the CRISPR/dCas9 system to activate expression of an anti-inflammatory and anti-apoptotic gene, such as heme oxygenase-1 (HO-1), to an optimal level to increase transplanted stem cell survival while also mitigating its cytotoxic effects due to lack of internal regulation, thus prolonging its effects within the ischemic myocardium leading to greater therapeutic benefit.
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Affiliation(s)
- Alexander Pan
- Vascular Biology Center, Department of Medicine, Medical College of Georgia/Georgia Regents University, 1459 Laney Walker Blvd, Augusta, GA 30912, USA
| | - Neal L Weintraub
- Vascular Biology Center, Department of Medicine, Medical College of Georgia/Georgia Regents University, 1459 Laney Walker Blvd, Augusta, GA 30912, USA
| | - Yaoliang Tang
- Vascular Biology Center, Department of Medicine, Medical College of Georgia/Georgia Regents University, 1459 Laney Walker Blvd, Augusta, GA 30912, USA.
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Zschemisch NH, Eisenblätter R, Rudolph C, Glage S, Dorsch M. Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm. SPRINGERPLUS 2014; 3:588. [PMID: 25332888 PMCID: PMC4197200 DOI: 10.1186/2193-1801-3-588] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Accepted: 09/22/2014] [Indexed: 01/02/2023]
Abstract
Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.
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Affiliation(s)
- Nils-Holger Zschemisch
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Regina Eisenblätter
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Cornelia Rudolph
- Institute for Molecular and Cellular Pathology, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Silke Glage
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
| | - Martina Dorsch
- Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany
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44
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Shao Y, Guan Y, Wang L, Qiu Z, Liu M, Chen Y, Wu L, Li Y, Ma X, Liu M, Li D. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 2014; 9:2493-512. [PMID: 25255092 DOI: 10.1038/nprot.2014.171] [Citation(s) in RCA: 152] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
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Affiliation(s)
- Yanjiao Shao
- 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2]
| | - Yuting Guan
- 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2]
| | - Liren Wang
- 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2]
| | - Zhongwei Qiu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Meizhen Liu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Yuting Chen
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Lijuan Wu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Yongmei Li
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Xueyun Ma
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Mingyao Liu
- 1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2] Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA
| | - Dali Li
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
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Hisamatsu S, Sakaue M, Takizawa A, Kato T, Kamoshita M, Ito J, Kashiwazaki N. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease. Anim Sci J 2014; 86:132-7. [DOI: 10.1111/asj.12259] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Accepted: 04/22/2014] [Indexed: 11/27/2022]
Affiliation(s)
- Shin Hisamatsu
- Department of Environmental Sciences; School of Life and Environmental Science; Azabu University; Sagamihara Japan
| | - Motoharu Sakaue
- Department of Anatomy II; Graduate School of Veterinary Sciences; Azabu University; Sagamihara Japan
- School of Veterinary Medicine; Azabu University; Sagamihara Japan
| | - Akiko Takizawa
- Graduate School of Medicine; Kyoto University; Kyoto Japan
| | - Tsubasa Kato
- Laboratory of Animal Reproduction; Graduate School of Veterinary Sciences; Azabu University; Sagamihara Japan
| | - Maki Kamoshita
- Laboratory of Animal Reproduction; Graduate School of Veterinary Sciences; Azabu University; Sagamihara Japan
| | - Junya Ito
- School of Veterinary Medicine; Azabu University; Sagamihara Japan
- Laboratory of Animal Reproduction; Graduate School of Veterinary Sciences; Azabu University; Sagamihara Japan
| | - Naomi Kashiwazaki
- School of Veterinary Medicine; Azabu University; Sagamihara Japan
- Laboratory of Animal Reproduction; Graduate School of Veterinary Sciences; Azabu University; Sagamihara Japan
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46
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The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment. Nat Commun 2014; 5:3818. [PMID: 24784029 PMCID: PMC4060806 DOI: 10.1038/ncomms4818] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 04/07/2014] [Indexed: 11/09/2022] Open
Abstract
Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16−/− ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16−/− ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.
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Savastano LE, Laurito SR, Fitt MR, Rasmussen JA, Gonzalez Polo V, Patterson SI. Sciatic nerve injury: A simple and subtle model for investigating many aspects of nervous system damage and recovery. J Neurosci Methods 2014; 227:166-80. [DOI: 10.1016/j.jneumeth.2014.01.020] [Citation(s) in RCA: 70] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2013] [Revised: 01/16/2014] [Accepted: 01/20/2014] [Indexed: 02/04/2023]
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48
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Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat Commun 2014; 4:2403. [PMID: 23985566 DOI: 10.1038/ncomms3403] [Citation(s) in RCA: 131] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Accepted: 08/05/2013] [Indexed: 12/18/2022] Open
Abstract
Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal, however, remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely, retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2, a downstream target of β-catenin, or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate.
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Ivics Z, Mátés L, Yau TY, Landa V, Zidek V, Bashir S, Hoffmann OI, Hiripi L, Garrels W, Kues WA, Bösze Z, Geurts A, Pravenec M, Rülicke T, Izsvák Z. Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 2014; 9:773-93. [PMID: 24625778 DOI: 10.1038/nprot.2014.008] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.
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Affiliation(s)
- Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Lajos Mátés
- Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
| | - Tien Yin Yau
- Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Vladimír Landa
- Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Vaclav Zidek
- Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Sanum Bashir
- Max Delbrück Center for Molecular Medicine, Berlin, Germany
| | | | | | - Wiebke Garrels
- Friedrich Loeffler Institut, Institut für Nutztiergenetik, Neustadt, Germany
| | - Wilfried A Kues
- Friedrich Loeffler Institut, Institut für Nutztiergenetik, Neustadt, Germany
| | | | - Aron Geurts
- Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
| | - Michal Pravenec
- Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Thomas Rülicke
- Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria
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50
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Hirabayashi M, Goto T, Tamura C, Sanbo M, Hara H, Hochi S. Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines. J Reprod Dev 2014; 60:78-82. [PMID: 24317016 PMCID: PMC3958585 DOI: 10.1262/jrd.2013-109] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2013] [Accepted: 11/11/2013] [Indexed: 11/20/2022] Open
Abstract
This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.
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Affiliation(s)
- Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 444-8787, Japan
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