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Davies B, Trelfa L, Rashbrook VS, Drydale E, Martin R, Bai B, Golebka J, Biggs DS, Channon KM, Bhattacharya S, Douglas G. Mutagenesis on a complex mouse genetic background by site-specific nucleases. Transgenic Res 2024; 33:415-426. [PMID: 39088185 PMCID: PMC11588839 DOI: 10.1007/s11248-024-00399-5] [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: 10/27/2023] [Accepted: 07/22/2024] [Indexed: 08/02/2024]
Abstract
Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.
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Affiliation(s)
- Benjamin Davies
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
- Francis Crick Institute, 1 Midland Road, London, UK
| | - Lucy Trelfa
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Victoria S Rashbrook
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Edward Drydale
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Rachel Martin
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Boyan Bai
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Jedrzej Golebka
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Daniel Stephen Biggs
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Keith M Channon
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
| | - Shoumo Bhattacharya
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK
| | - Gillian Douglas
- Division of Cardiovascular Medicine, Radcliffe Department of Medicine, BHF Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK.
- Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK.
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2
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Elizalde MJ, Gorelick DA. Mechanistic toxicology in light of genetic compensation. Toxicol Sci 2023; 197:kfad113. [PMID: 37941503 PMCID: PMC10823772 DOI: 10.1093/toxsci/kfad113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2023] Open
Abstract
Mechanistic toxicology seeks to identify the molecular and cellular mechanisms by which toxicants exert their deleterious effects. One powerful approach is to generate mutations in genes that respond to a particular toxicant, and then test how such mutations change the effects of the toxicant. CRISPR is a rapid and versatile approach to generate mutations in cultured cells and in animal models. Many studies use CRISPR to generate short insertions or deletions in a target gene and then assume that the resulting mutation, such as a premature termination codon, causes a loss of functional protein. However, recent studies demonstrate that this assumption is flawed. Cells can compensate for short insertion and deletion mutations, leading toxicologists to draw erroneous conclusions from mutant studies. In this review, we will discuss mechanisms by which a mutation in one gene may be rescued by compensatory activity. We will discuss how CRISPR insertion and deletion mutations are susceptible to compensation by transcriptional adaptation, alternative splicing, and rescue by maternally derived gene products. We will review evidence that measuring levels of messenger RNA transcribed from a mutated gene is an unreliable indicator of the severity of the mutation. Finally, we provide guidelines for using CRISPR to generate mutations that avoid compensation.
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Affiliation(s)
- Mary Jane Elizalde
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
| | - Daniel A Gorelick
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
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Phan HTL, Kim K, Lee H, Seong JK. Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications. Genes (Basel) 2023; 14:483. [PMID: 36833410 PMCID: PMC9957140 DOI: 10.3390/genes14020483] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/09/2023] [Accepted: 02/10/2023] [Indexed: 02/17/2023] Open
Abstract
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are widely accepted because of their diversity and enormous potential for targeted genomic modifications in eukaryotes and other animals. Moreover, rapid advances in genome editing tools have accelerated the ability to produce various genetically modified animal models for studying human diseases. Given the advances in gene editing tools, these animal models are gradually evolving toward mimicking human diseases through the introduction of human pathogenic mutations in their genome rather than the conventional gene knockout. In the present review, we summarize the current progress in and discuss the prospects for developing mouse models of human diseases and their therapeutic applications based on advances in the study of programmable nucleases.
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Affiliation(s)
- Hong Thi Lam Phan
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Kyoungmi Kim
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
- Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Ho Lee
- Graduate School of Cancer Science and Policy, National Cancer Center, Goyang 10408, Republic of Korea
| | - Je Kyung Seong
- Korea Mouse Phenotyping Center, Seoul National University, Seoul 08826, Republic of Korea
- Laboratory of Developmental Biology and Genomics, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
- Interdisciplinary Program for Bioinformatics, Program for Cancer Biology, BIO-MAX/N-Bio Institute, Seoul National University, Seoul 08826, Republic of Korea
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Eksi YE, Sanlioglu AD, Akkaya B, Ozturk BE, Sanlioglu S. Genome engineering and disease modeling via programmable nucleases for insulin gene therapy; promises of CRISPR/Cas9 technology. World J Stem Cells 2021; 13:485-502. [PMID: 34249224 PMCID: PMC8246254 DOI: 10.4252/wjsc.v13.i6.485] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 04/02/2021] [Accepted: 06/16/2021] [Indexed: 02/06/2023] Open
Abstract
Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change, insert, or remove a genomic sequence of interest. These advanced molecular tools include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and RNA-guided engineered nucleases (RGENs), which create double-strand breaks at specific target sites in the genome, and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism. A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype, without the need for the reengineering of the specific enzyme when targeting different sequences. CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function. RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes, as summarized and exemplified in this manuscript.
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Affiliation(s)
- Yunus E Eksi
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Ahter D Sanlioglu
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Bahar Akkaya
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Bilge Esin Ozturk
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, United States
| | - Salih Sanlioglu
- Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey.
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CRISPR-Cas systems for genome editing of mammalian cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:15-30. [PMID: 34127192 DOI: 10.1016/bs.pmbts.2021.01.011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
In the past decade, ZFNs and TALENs have been used for targeted genome engineering and have gained scientific attention. It has demonstrated huge potential for gene knockout, knock-in, and indels in desired locations of genomes to understand molecular mechanism of diseases and also discover therapy. However, both the genome engineering techniques are still suffering from design, screening and validation in cell and higher organisms. CRISPR-Cas9 is a rapid, simple, specific, and versatile technology and it has been applied in many organisms including mammalian cells. CRISPR-Cas9 has been used for animal models to modify animal cells for understanding human disease for novel drug discovery and therapy. Additionally, base editing has also been discussed herewith for conversion of C/G-to-T/A or A/T-to-G/C without DNA cleavage or donor DNA templates for correcting mutations or altering gene functions. In this chapter, we highlight CRISPR-Cas9 and base editing for desired genome editing in mammalian cells for a better understanding of molecular mechanisms, and biotechnological and therapeutic applications.
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Singh V. An introduction to CRISPR-Cas systems for reprogramming the genome of mammalian cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:1-13. [PMID: 34127190 DOI: 10.1016/bs.pmbts.2021.01.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
In the past few decades, it has been possible to introduce unprecedented mutations in genes of the mammalian cells owing to the development of advanced technologies/methods/assays. Sometimes, these mutations occurring at the cellular level may even cost the life of organisms. A number of diseases in mammals have shown to leave some serious impact on their lives. There are no drugs or medicines available in market for the correction or repair of these mutated genes in order to reverse gene function. A pressing need therefore arises to develop a next generation technology that cannot just corrects gene mutations but also restores gene function. Recent advances in CRISPR-Cas9 technology play a key role for correction of defective genes in wide range of mammalian cells. This chapter highlights CRISPR-Cas systems for basic, biomedical, biotechnological and therapeutic applications.
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Affiliation(s)
- Vijai Singh
- Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India.
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7
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Lanigan TM, Kopera HC, Saunders TL. Principles of Genetic Engineering. Genes (Basel) 2020; 11:E291. [PMID: 32164255 PMCID: PMC7140808 DOI: 10.3390/genes11030291] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 02/28/2020] [Accepted: 03/06/2020] [Indexed: 12/12/2022] Open
Abstract
Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.
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Affiliation(s)
- Thomas M. Lanigan
- Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA; (T.M.L.); (H.C.K.)
- Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Huira C. Kopera
- Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA; (T.M.L.); (H.C.K.)
- Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
| | - Thomas L. Saunders
- Biomedical Research Core Facilities, Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109, USA
- Department of Internal Medicine, Division of Genetic Medicine, University of Michigan, Ann Arbor, MI 48109, USA
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8
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Xu J, Zhou C, Foo KS, Yang R, Xiao Y, Bylund K, Sahara M, Chien KR. Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors. Stem Cells 2020; 38:741-755. [PMID: 32129551 PMCID: PMC7891398 DOI: 10.1002/stem.3168] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2019] [Revised: 01/15/2020] [Accepted: 01/29/2020] [Indexed: 12/20/2022]
Abstract
Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two‐stage genome‐wide CRISPR‐knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA‐Seq profiling of the ZIC2‐mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA‐seq analysis showed the ZIC2 mutants affected the apelin receptor‐related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome‐wide unbiased CRISPR‐knockout screen to identify the key steps in human mesoderm precursor cell‐ and heart progenitor cell‐fate determination during in vitro hPSC cardiogenesis.
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Affiliation(s)
- Jiejia Xu
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Chikai Zhou
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Kylie S Foo
- Department of Medicine, Karolinska Institutet, Huddinge, Sweden
| | - Ran Yang
- Department of Medicine, Karolinska Institutet, Huddinge, Sweden
| | - Yao Xiao
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Kristine Bylund
- Department of Medicine, Karolinska Institutet, Huddinge, Sweden
| | - Makoto Sahara
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.,Department of Medicine, Karolinska Institutet, Huddinge, Sweden
| | - Kenneth R Chien
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.,Department of Medicine, Karolinska Institutet, Huddinge, Sweden
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9
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Abstract
A transgenic mouse carries within its genome an artificial DNA construct (transgene) that is deliberately introduced by an experimentalist. These animals are widely used to understand gene function and protein function. When addressing the history of transgenic mouse technology, it is apparent that a number of basic science research areas laid the groundwork for success. These include reproductive science, genetics and molecular biology, and micromanipulation and microscopy equipment. From reproductive physiology came applications on how to optimize mouse breeding, how to superovulate mice to produce zygotes for DNA microinjection or preimplantation embryos for combination with embryonic stem (ES) cells, and how to return zygotes and embryos to a pseudopregnant surrogate dam for gestation and birth. From developmental biology, it was learned how to micromanipulate embryos for morula aggregation and blastocyst microinjection and how to establish germline competent ES cells. From genetics came the foundational principles governing the inheritance of genes, the interactions of gene products, and an understanding of the phenotypic consequences of genetic mutations. From molecular biology came a panoply of tools and reagents that are used to clone DNA transgenes, to detect the presence of transgenes, to assess gene expression by measuring transcription, and to detect proteins in cells and tissues. Technical advances in light microscopes, micromanipulators, micropipette pullers, and ancillary equipment made it possible for experimentalists to insert thin glass needles into zygotes or embryos under controlled conditions to inject DNA solutions or ES cells. To fully discuss the breadth of contributions of these numerous scientific disciplines to a comprehensive history of transgenic science is beyond the scope of this work. Examples will be used to illustrate scientific developments central to the foundation of transgenic technology and that are in use today.
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Affiliation(s)
- Thomas L Saunders
- Transgenic Animal Model Core, University of Michigan Medical School, Ann Arbor, MI, USA.
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
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A Requirement for Zic2 in the Regulation of Nodal Expression Underlies the Establishment of Left-Sided Identity. Sci Rep 2018; 8:10439. [PMID: 29992973 PMCID: PMC6041270 DOI: 10.1038/s41598-018-28714-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Accepted: 06/25/2018] [Indexed: 12/26/2022] Open
Abstract
ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.
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11
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Diamand KEM, Barratt KS, Arkell RM. Overview of Rodent Zic Genes. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018; 1046:179-207. [PMID: 29442323 DOI: 10.1007/978-981-10-7311-3_10] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The five murine Zic genes encode multifunctional transcriptional regulator proteins important for a large number of processes during embryonic development. The genes and proteins are highly conserved with respect to the orthologous human genes, an attribute evidently mirrored by functional conservation, since the murine and human genes mutate to give the same phenotypes. Each ZIC protein contains a zinc finger domain that participates in both protein-DNA and protein-protein interactions. The ZIC proteins are capable of interacting with the key transcriptional mediators of the SHH, WNT and NODAL signalling pathways as well as with components of the transcriptional machinery and chromatin-modifying complexes. It is possible that this diverse range of protein partners underlies characteristics uncovered by mutagenesis and phenotyping of the murine Zic genes. These features include redundant and unique roles for ZIC proteins, regulatory interdependencies amongst family members and pleiotropic Zic gene function. Future investigations into the complex nature of the Zic gene family activity should be facilitated by recent advances in genome engineering and functional genomics.
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Affiliation(s)
- Koula E M Diamand
- Early Mammalian Development Laboratory, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
| | - Kristen S Barratt
- Early Mammalian Development Laboratory, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
| | - Ruth M Arkell
- Early Mammalian Development Laboratory, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.
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Hanssen LLP, Kassouf MT, Oudelaar AM, Biggs D, Preece C, Downes DJ, Gosden M, Sharpe JA, Sloane-Stanley JA, Hughes JR, Davies B, Higgs DR. Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo. Nat Cell Biol 2017; 19:952-961. [PMID: 28737770 PMCID: PMC5540176 DOI: 10.1038/ncb3573] [Citation(s) in RCA: 156] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2017] [Accepted: 06/15/2017] [Indexed: 12/16/2022]
Abstract
The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
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Affiliation(s)
- Lars L P Hanssen
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
- The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK
| | - Mira T Kassouf
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | - A Marieke Oudelaar
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | - Daniel Biggs
- The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK
| | - Chris Preece
- The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK
| | - Damien J Downes
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | - Matthew Gosden
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | - Jacqueline A Sharpe
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | | | - Jim R Hughes
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
| | - Benjamin Davies
- The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK
| | - Douglas R Higgs
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK
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13
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Freeburg SH, Engelbrecht E, Powell WH. Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type. Toxicol Sci 2016; 155:337-347. [PMID: 27994169 DOI: 10.1093/toxsci/kfw212] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Gene duplication confers genetic redundancy that can facilitate subfunctionalization, the partitioning of ancestral functions between paralogs. We capitalize on a recent genome duplication in Xenopus laevis (African clawed frog) to interrogate possible functional differentiation between alloalleles of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates toxicity of dioxin-like compounds and plays a role in the physiology and development of the cardiovascular, hepatic, and immune systems in vertebrates. X. laevis has 2 AHR genes, AHR1α and AHR1β To test the hypothesis that the encoded proteins exhibit different molecular functions, we used TALENs in XLK-WG cells, generating mutant lines lacking functional versions of each AHR and measuring the transcriptional responsiveness of several target genes to the toxic xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the candidate endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). Mutation of either AHR1α or AHR1β reduced TCDD induction of the canonical AHR target, Cytochrome P4501A6, by 75%, despite the much lower abundance of AHR1β in wild-type cells. More modestly induced target genes, encoding aryl hydrocarbon receptor repressor (AHRR), spectrin repeat-containing nuclear envelope protein 1 (SYNE-1), and gap junction protein gamma 1 (GJC1), were regulated solely by AHR1α. AHR1β was responsible for CYP1A6 induction by FICZ, while AHR1α mediated FICZ induction of AHRR We conclude that AHR1α and AHR1β have distinct transcriptional functions in response to specific agonists, even within a single cell type. Functional analysis of frog AHR paralogs advances the understanding of AHR evolution and as well as the use of frog models of developmental toxicology such as FETAX.
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Affiliation(s)
| | | | - Wade H Powell
- Biology Department, Kenyon College, Gambier, Ohio 43022
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14
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Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer. Sci Rep 2016; 6:33675. [PMID: 27654750 PMCID: PMC5031972 DOI: 10.1038/srep33675] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2016] [Accepted: 08/31/2016] [Indexed: 01/14/2023] Open
Abstract
Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.
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15
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Nanjidsuren T, Park CW, Sim BW, Kim SU, Chang KT, Kang MH, Min KS. GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting. Anim Biotechnol 2016; 27:223-30. [DOI: 10.1080/10495398.2016.1176032] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Affiliation(s)
- Tsevelmaa Nanjidsuren
- Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong, Republic of Korea
| | - Chae-Won Park
- Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong, Republic of Korea
| | - Bo-Woong Sim
- National Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea
| | - Sun-Uk Kim
- National Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea
| | - Kyu-Tae Chang
- National Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea
| | - Myung-Hwa Kang
- Department of Food and Nutrition, Hoseo University, Asan, Republic of Korea
| | - Kwan-Sik Min
- Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong, Republic of Korea
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16
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Abstract
Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.
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Affiliation(s)
- Benedikt Wefers
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany.
- Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE), 81377, Munich, Germany.
| | - Christina Brandl
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany.
- Technische Universität München, 85350, Freising-Weihenstephan, Germany.
| | - Oskar Ortiz
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany.
| | - Wolfgang Wurst
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany.
- Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE), 81377, Munich, Germany.
- Technische Universität München, 85350, Freising-Weihenstephan, Germany.
- Max Planck Institute of Psychiatry, 80804, Munich, Germany.
- Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, 81377, Munich, Germany.
| | - Ralf Kühn
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764, Neuherberg, Germany.
- Technische Universität München, 85350, Freising-Weihenstephan, Germany.
- Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany.
- Berlin Institute of Health, Berlin, Germany.
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17
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Zhao X, Ni W, Chen C, Sai W, Qiao J, Sheng J, Zhang H, Li G, Wang D, Hu S. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2016; 29:413-8. [PMID: 26950874 PMCID: PMC4811794 DOI: 10.5713/ajas.15.0041] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Revised: 04/22/2015] [Accepted: 07/17/2015] [Indexed: 11/27/2022]
Abstract
Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.
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Affiliation(s)
- Xinxia Zhao
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
- College of Life Sciences, Shihezi University, Shihezi, Xinjiang, 832003,
China
| | - Wei Ni
- College of Life Sciences, Shihezi University, Shihezi, Xinjiang, 832003,
China
| | - Chuangfu Chen
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Wujiafu Sai
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Jun Qiao
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Jingliang Sheng
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Hui Zhang
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Guozhong Li
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003,
China
| | - Dawei Wang
- College of Life Sciences, Shihezi University, Shihezi, Xinjiang, 832003,
China
| | - Shengwei Hu
- College of Life Sciences, Shihezi University, Shihezi, Xinjiang, 832003,
China
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18
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Cellular Engineering and Disease Modeling with Gene-Editing Nucleases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_12] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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19
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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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20
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Proetzel G, Wiles MV, Roopenian DC. Genetically engineered humanized mouse models for preclinical antibody studies. BioDrugs 2015; 28:171-80. [PMID: 24150980 DOI: 10.1007/s40259-013-0071-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies.
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21
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Beil J, Buch T. Generation of bacterial artificial chromosome (BAC) transgenic mice. Methods Mol Biol 2015; 1194:157-69. [PMID: 25064102 DOI: 10.1007/978-1-4939-1215-5_8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/10/2023]
Abstract
Transgenic mice are among the most helpful tools to study the role of genes in physiological conditions. In this protocol, we describe the generation of bacterial artificial chromosome (BACs) constructs, which are used to express a gene of interest under a particular promoter. BACs as driver of transgenes have the advantage that a characterization of transcriptional control elements is unnecessary and the construct's size usually reduces position effects from random integration. In the following, we firstly explain in detail the amplification of the BAC, the generation of the targeting construct as well as the recombination by ET-cloning, and the analysis of the recombined clones by Southern blot analysis. Finally, we also describe the preparation of the BACs for oocyte injection. In total, the construction of such BAC transgenes needs around 6-8 weeks.
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Affiliation(s)
- Jane Beil
- Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Trogerstr. 30, 81675, Munich, Germany
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22
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Matsubara Y, Kato T, Kashimada K, Tanaka H, Zhi Z, Ichinose S, Mizutani S, Morio T, Chiba T, Ito Y, Saga Y, Takada S, Asahara H. TALEN-Mediated Gene Disruption on Y Chromosome Reveals Critical Role of EIF2S3Y in Mouse Spermatogenesis. Stem Cells Dev 2015; 24:1164-70. [PMID: 25579647 DOI: 10.1089/scd.2014.0466] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
The Y chromosome plays a critical role in spermatogenesis. Formerly, it had been difficult to generate knockout mice with specific Y chromosome mutations using conventional gene-targeting strategies. Recently, a transcription activator-like effector nuclease (TALEN) was successfully used for editing a mouse Y chromosome-linked gene. Here, we report the generation of a mouse model with a mutation in EIF2S3Y, a Y chromosome-linked gene, and analysis of its phenotype. The mouse carrying a targeted mutation of EIF2S3Y was infertile and had hypoplastic testes. Histological and electron microscopic analyses showed that differentiation of spermatogonia was arrested at the stage of spermatogonial stem cells (undifferentiated spermatogonia) and that the progression of spermatogenesis was interrupted, resulting in azoospermia. Using TALEN, we verified that EIF2S3Y performs a key function in differentiation of spermatogonial stem cells.
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Affiliation(s)
- Yohei Matsubara
- 1 Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University , Tokyo, Japan
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23
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Nakagawa Y, Sakuma T, Nakagata N, Yamasaki S, Takeda N, Ohmuraya M, Yamamoto T. Application of oocyte cryopreservation technology in TALEN-mediated mouse genome editing. Exp Anim 2015; 63:349-55. [PMID: 25077765 PMCID: PMC4206739 DOI: 10.1538/expanim.63.349] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Reproductive engineering techniques, such as in vitro fertilization
(IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation,
reproduction, and transportation of genetically engineered mice. However, it has not yet
been elucidated whether these techniques can be applied for the generation of
genome-edited mice using engineered nucleases such as transcription activator-like
effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes
fertilized in vitro using frozen sperm for TALEN-mediated genome editing
in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen),
fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the
source of oocytes used for TALEN injection; we found that fertilized oocytes created under
all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we
investigated whether the ages in weeks of parental female mice can affect the efficiency
of gene modification, by comparing 5-week-old and 8–12-week-old mice as the source of
oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was
consistently 95–100% when either 5-week-old or 8–12-week-old mice were used with or
without freezing the oocytes. Thus, our report describes the availability of freeze-thawed
oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome
editing, thus boosting the convenience of such innovative gene targeting strategies.
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Affiliation(s)
- Yoshiko Nakagawa
- Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
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24
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Sommer D, Peters AE, Baumgart AK, Beyer M. TALEN-mediated genome engineering to generate targeted mice. Chromosome Res 2015; 23:43-55. [DOI: 10.1007/s10577-014-9457-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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25
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Nakagawa Y, Yamamoto T, Suzuki KI, Araki K, Takeda N, Ohmuraya M, Sakuma T. Screening methods to identify TALEN-mediated knockout mice. Exp Anim 2014; 63:79-84. [PMID: 24521866 PMCID: PMC4160933 DOI: 10.1538/expanim.63.79] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Genome editing with site-specific nucleases, such as zinc-finger nucleases or
transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such
as the CRISPR/Cas (clustered regularly interspaced short palindromic
repeats/CRISPR-associated) system, is becoming the new standard for targeted genome
modification in various organisms. Application of these techniques to the manufacture of
knockout mice would be greatly aided by simple and easy methods for genotyping of mutant
and wild-type pups among litters. However, there are no detailed or comparative reports
concerning the identification of mutant mice generated using genome editing technologies.
Here, we genotyped TALEN-derived enhanced green fluorescent protein
(eGFP) knockout mice using a combination of approaches, including
fluorescence observation, heteroduplex mobility assay, restriction fragment length
polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced
mutations differed among these methods, and we therefore concluded that combinatorial
testing is necessary for the screening and determination of mutant genotypes. Since the
analytical methods tested can be carried out without specialized equipment, costly
reagents and/or sophisticated protocols, our report should be of interest to a broad range
of researchers who are considering the application of genome editing technologies in
various organisms.
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Affiliation(s)
- Yoshiko Nakagawa
- Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
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26
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Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov 2014; 13:759-80. [PMID: 25233993 DOI: 10.1038/nrd4278] [Citation(s) in RCA: 1522] [Impact Index Per Article: 138.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.
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Affiliation(s)
- Ugur Sahin
- 1] TRON Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Langenbeckstrasse 1, 55131 Mainz, Germany. [2] BioNTech Corporation, An der Goldgrube 12, 55131 Mainz, Germany
| | - Katalin Karikó
- 1] BioNTech Corporation, An der Goldgrube 12, 55131 Mainz, Germany. [2] Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
| | - Özlem Türeci
- TRON Translational Oncology at the University Medical Center of the Johannes Gutenberg University, Langenbeckstrasse 1, 55131 Mainz, Germany
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27
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Ni W, Qiao J, Hu S, Zhao X, Regouski M, Yang M, Polejaeva IA, Chen C. Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 2014; 9:e106718. [PMID: 25188313 PMCID: PMC4154755 DOI: 10.1371/journal.pone.0106718] [Citation(s) in RCA: 157] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2014] [Accepted: 08/10/2014] [Indexed: 11/19/2022] Open
Abstract
The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.
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Affiliation(s)
- Wei Ni
- College of Life Sciences, Shihezi University, Shihezi, Xinjiang, China
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
| | - Jun Qiao
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China
| | - Shengwei Hu
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
- * E-mail: (SH); (IP); (CC)
| | - Xinxia Zhao
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China
| | - Misha Regouski
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
| | - Min Yang
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
| | - Irina A. Polejaeva
- Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
- * E-mail: (SH); (IP); (CC)
| | - Chuangfu Chen
- College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China
- * E-mail: (SH); (IP); (CC)
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28
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Abstract
Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.
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29
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Yasue A, Mitsui SN, Watanabe T, Sakuma T, Oyadomari S, Yamamoto T, Noji S, Mito T, Tanaka E. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems. Sci Rep 2014; 4:5705. [PMID: 25027812 PMCID: PMC4099983 DOI: 10.1038/srep05705] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2013] [Accepted: 06/27/2014] [Indexed: 02/06/2023] Open
Abstract
Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.
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Affiliation(s)
- Akihiro Yasue
- Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
| | - Silvia Naomi Mitsui
- Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
| | - Takahito Watanabe
- Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan
| | - Tetsushi Sakuma
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
| | - Seiichi Oyadomari
- Division of Molecular Biology, Institute for Genome Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Takashi Yamamoto
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan
| | - Sumihare Noji
- Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan
| | - Taro Mito
- Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan
| | - Eiji Tanaka
- Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
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30
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Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae 2014; 6:19-40. [PMID: 25349712 PMCID: PMC4207558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn't enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.
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Affiliation(s)
- A. A. Nemudryi
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090
- Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055
| | - K. R. Valetdinova
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090
- Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055
- Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
| | - S. P. Medvedev
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090
- Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055
- Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
| | - S. M. Zakian
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090
- Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055
- Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
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Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae 2014; 85:209-18. [PMID: 25349712 DOI: 10.1007/s11103-014-0188-7] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2014] [Accepted: 03/05/2014] [Indexed: 05/07/2023] Open
Abstract
Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn't enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.
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Affiliation(s)
- A A Nemudryi
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090 ; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090 ; Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055
| | - K R Valetdinova
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090 ; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090 ; Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055 ; Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
| | - S P Medvedev
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090 ; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090 ; Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055 ; Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
| | - S M Zakian
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 10, Novosibirsk, Russia, 630090 ; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Prosp., 8, Novosibirsk, Russia, 630090 ; Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Ministry of Health of the Russian Federation, Rechkunovskaya Str., 15, Novosibirsk, Russia, 630055 ; Novosibirsk State University, Pirogova Str., 2, Novosibirsk, Russia, 630090
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Zantke J, Bannister S, Rajan VBV, Raible F, Tessmar-Raible K. Genetic and genomic tools for the marine annelid Platynereis dumerilii. Genetics 2014; 197:19-31. [PMID: 24807110 PMCID: PMC4012478 DOI: 10.1534/genetics.112.148254] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2013] [Accepted: 02/17/2014] [Indexed: 01/27/2023] Open
Abstract
The bristle worm Platynereis dumerilii displays many interesting biological characteristics. These include its reproductive timing, which is synchronized to the moon phase, its regenerative capacity that is hormonally controlled, and a slow rate of evolution, which permits analyses of ancestral genes and cell types. As a marine annelid, Platynereis is also representative of the marine ecosystem, as well as one of the three large animal subphyla, the Lophotrochozoa. Here, we provide an overview of the molecular resources, functional techniques, and behavioral assays that have recently been established for the bristle worm. This combination of tools now places Platynereis in an excellent position to advance research at the frontiers of neurobiology, chronobiology, evo-devo, and marine biology.
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Affiliation(s)
- Juliane Zantke
- Max F. Perutz Laboratories
- Research Platform Marine Rhythms of Life, University of Vienna 1030 Vienna, Austria
| | - Stephanie Bannister
- Max F. Perutz Laboratories
- Research Platform Marine Rhythms of Life, University of Vienna 1030 Vienna, Austria
| | - Vinoth Babu Veedin Rajan
- Max F. Perutz Laboratories
- Research Platform Marine Rhythms of Life, University of Vienna 1030 Vienna, Austria
| | - Florian Raible
- Max F. Perutz Laboratories
- Research Platform Marine Rhythms of Life, University of Vienna 1030 Vienna, Austria
| | - Kristin Tessmar-Raible
- Max F. Perutz Laboratories
- Research Platform Marine Rhythms of Life, University of Vienna 1030 Vienna, Austria
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Wijshake T, Baker DJ, van de Sluis B. Endonucleases: new tools to edit the mouse genome. Biochim Biophys Acta Mol Basis Dis 2014; 1842:1942-1950. [PMID: 24794718 DOI: 10.1016/j.bbadis.2014.04.020] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 04/16/2014] [Accepted: 04/18/2014] [Indexed: 12/26/2022]
Abstract
Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function.
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Affiliation(s)
- Tobias Wijshake
- Molecular Genetics, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
| | - Darren J Baker
- Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905, USA
| | - Bart van de Sluis
- Molecular Genetics, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
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TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii. Genetics 2014; 197:77-89. [PMID: 24653002 PMCID: PMC4012502 DOI: 10.1534/genetics.113.161091] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Platynereis dumerilii is a marine polychaete and an established model system for studies of evolution and development. Platynereis is also a re-emerging model for studying the molecular basis of circalunar reproductive timing: a biological phenomenon observed in many marine species. While gene expression studies have provided new insight into patterns of gene regulation, a lack of reverse genetic tools has so far limited the depth of functional analyses in this species. To address this need, we established customized transcriptional activator-like effector nucleases (TALENs) as a tool to engineer targeted modifications in Platynereis genes. By adapting a workflow of TALEN construction protocols and mutation screening approaches for use in Platynereis, we engineered frameshift mutations in three endogenous Platynereis genes. We confirmed that such mutations are heritable, demonstrating that TALENs can be used to generate homozygous knockout lines in P. dumerilii. This is the first use of TALENs for generating genetic knockout mutations in an annelid model. These tools not only open the door for detailed in vivo functional analyses, but also can facilitate further technical development, such as targeted genome editing.
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Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes. Mol Cell Biol 2014; 34:1649-58. [PMID: 24567370 DOI: 10.1128/mcb.00023-14] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.
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36
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Generation of TALEN-mediated GRdim knock-in rats by homologous recombination. PLoS One 2014; 9:e88146. [PMID: 24523878 PMCID: PMC3921256 DOI: 10.1371/journal.pone.0088146] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2013] [Accepted: 01/04/2014] [Indexed: 11/19/2022] Open
Abstract
Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.
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Wefers B, Ortiz O, Wurst W, Kühn R. Generation of targeted mouse mutants by embryo microinjection of TALENs. Methods 2014; 69:94-101. [PMID: 24418396 DOI: 10.1016/j.ymeth.2014.01.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 01/01/2014] [Accepted: 01/02/2014] [Indexed: 12/26/2022] Open
Abstract
Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.
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Affiliation(s)
- Benedikt Wefers
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany.
| | - Oskar Ortiz
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany.
| | - Wolfgang Wurst
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany; Technische Universität München, 85350 Freising-Weihenstephan, Germany; Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE), 80336 Munich, Germany; Max Planck Institute of Psychiatry, 80804 Munich, Germany.
| | - Ralf Kühn
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany; Technische Universität München, 85350 Freising-Weihenstephan, Germany.
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Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P, Chen M, Zhou Q. A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 2014; 9:e84893. [PMID: 24416307 PMCID: PMC3885652 DOI: 10.1371/journal.pone.0084893] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2013] [Accepted: 11/20/2013] [Indexed: 02/05/2023] Open
Abstract
The transcription activator-like effector nucleases (TALENs) strategy has been widely used to delete and mutate genes in vitro. This strategy has begun to be used for in vivo systemic gene manipulation, but not in an organ-specific manner. In this study, we developed a modified, highly efficient TALEN strategy using a dual-fluorescence reporter. We used this modified strategy and, within 5 weeks, we successfully generated kidney proximal tubule-specific gene Ttc36 homozygous knockout mice. Unilateral nephrectomy was performed on the 6-week-old founders (F0) to identify the knockout genotype prior to the birth of the offspring. This strategy was found to have little effect on reproduction in the knockout mice and inheritability of the knockout genotypes. The modified TALEN knockout strategy in combination with unilateral nephrectomy can be readily used for studies of gene function in kidney development and diseases.
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Affiliation(s)
- Yunhong Liu
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Xiaoyan Lv
- Department of Dermatology, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Ruizhi Tan
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Tianming Liu
- Lab of Molecular Nephrology, Chongqing Medical University, Chongqing, China
| | - Tielin Chen
- State Key Laboratory of Biotherapy and Cancer Center, Sichuan University, Chengdu, Sichuan, China
| | - Mi Li
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Yuhang Liu
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Fang Nie
- Lab of Molecular Nephrology, Chongqing Medical University, Chongqing, China
| | - Xiaoyan Wang
- Lab of Molecular Nephrology, Chongqing Medical University, Chongqing, China
| | - Puhui Zhou
- Lab of Molecular Nephrology, Chongqing Medical University, Chongqing, China
- * E-mail: (PZ); (QZ)
| | - Mianzhi Chen
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
| | - Qin Zhou
- Core Facility of Genetically Engineered Mice, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China
- * E-mail: (PZ); (QZ)
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39
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Jones JM, Meisler MH. Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a. Genesis 2013; 52:141-8. [PMID: 24288358 DOI: 10.1002/dvg.22731] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2013] [Revised: 11/21/2013] [Accepted: 11/25/2013] [Indexed: 01/09/2023]
Abstract
To evaluate the efficiency of TALEN technology for introducing mutations into the mouse genome we targeted Scn8a, a member of a multigene family with nine closely related paralogs. Our goal was to generate a model of early onset epileptic encephalopathy by introduction of the Scn8a missense mutation p.Asn1768Asp. We used a pair of TALENs that were highly active in transfected cells. The targeting template for homologous recombination contained a 4 kb genomic fragment. Microinjection of TALENs with the targeting construct into the pronucleus of 350 fertilized mouse eggs generated 67 live-born potential founders, of which 5 were heterozygous for the pathogenic mutation, a yield of 7% correctly targeted mice. Twenty-four mice carried one or two Scn8a indels, including 12 frameshift mutations and the novel amino acid deletion p.Asn1759del. Nine off-site mutations in the paralogs sodium channel genes Scn5a and Scn4a were identified. The data demonstrate the feasibility and efficiency of targeting members of multigene families using TALENs. The Scn8a(tm) (1768DMm) mouse model will be useful for investigation of the pathogenesis and therapy of early onset seizure disorders.
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Affiliation(s)
- Julie M Jones
- Department of Human Genetics, University of Michigan, Ann Arbor, Michigan
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Abstract
Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.
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Affiliation(s)
- Jarryd M Campbell
- Center for Translational Science Activities, Mayo Clinic, Rochester, MN 55905, USA.
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41
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Aida T, Imahashi R, Tanaka K. Translating human genetics into mouse: The impact of ultra-rapidin vivogenome editing. Dev Growth Differ 2013; 56:34-45. [DOI: 10.1111/dgd.12101] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2013] [Revised: 10/09/2013] [Accepted: 10/10/2013] [Indexed: 12/26/2022]
Affiliation(s)
- Tomomi Aida
- Laboratory of Molecular Neuroscience; Medical Research Institute; Tokyo Medical and Dental University; 1-5-45 Yushima Bunkyo-Ku Tokyo, 113-8510 Japan
| | - Risa Imahashi
- Laboratory of Molecular Neuroscience; Medical Research Institute; Tokyo Medical and Dental University; 1-5-45 Yushima Bunkyo-Ku Tokyo, 113-8510 Japan
| | - Kohichi Tanaka
- Laboratory of Molecular Neuroscience; Medical Research Institute; Tokyo Medical and Dental University; 1-5-45 Yushima Bunkyo-Ku Tokyo, 113-8510 Japan
- The Center for Brain Integration Research; Tokyo Medical and Dental University; 1-5-45 Yushima Bunkyo-Ku Tokyo, 113-8510 Japan
- JST; CREST; 4-1-8 Honcho Kawaguchi-shi Saitama 332-0012 Japan
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42
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Zhang Z, Zhang S, Huang X, Orwig KE, Sheng Y. Rapid assembly of customized TALENs into multiple delivery systems. PLoS One 2013; 8:e80281. [PMID: 24244669 PMCID: PMC3820630 DOI: 10.1371/journal.pone.0080281] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2013] [Accepted: 10/11/2013] [Indexed: 11/18/2022] Open
Abstract
Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway(®) Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated "user friendly" TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains.
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Affiliation(s)
- Zhengxing Zhang
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America
| | - Siliang Zhang
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America
| | - Xin Huang
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America
- Women’s Cancer Research Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America
| | - Kyle E. Orwig
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America
| | - Yi Sheng
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute and Foundation, Pittsburgh, Pennsylvania, United States of America
- * E-mail:
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43
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Zhang C, Wang L, Ren G, Li Z, Ren C, Zhang T, Xu K, Zhang Z. Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases. Mol Biol Rep 2013; 41:209-15. [PMID: 24197697 DOI: 10.1007/s11033-013-2853-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2012] [Accepted: 10/30/2013] [Indexed: 10/26/2022]
Abstract
Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.
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Affiliation(s)
- Cunfang Zhang
- College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Xianyang, 712100, Shaanxi, People's Republic of China
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Kato T, Miyata K, Sonobe M, Yamashita S, Tamano M, Miura K, Kanai Y, Miyamoto S, Sakuma T, Yamamoto T, Inui M, Kikusui T, Asahara H, Takada S. Production of Sry knockout mouse using TALEN via oocyte injection. Sci Rep 2013; 3:3136. [PMID: 24190364 PMCID: PMC3817445 DOI: 10.1038/srep03136] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2013] [Accepted: 10/16/2013] [Indexed: 12/02/2022] Open
Abstract
Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.
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Affiliation(s)
- Tomoko Kato
- 1] Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan [2]
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Wefers B, Panda SK, Ortiz O, Brandl C, Hensler S, Hansen J, Wurst W, Kühn R. Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA. Nat Protoc 2013; 8:2355-79. [PMID: 24177293 DOI: 10.1038/nprot.2013.142] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.
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Affiliation(s)
- Benedikt Wefers
- Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany
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Takada S, Sato T, Ito Y, Yamashita S, Kato T, Kawasumi M, Kanai-Azuma M, Igarashi A, Kato T, Tamano M, Asahara H. Targeted gene deletion of miRNAs in mice by TALEN system. PLoS One 2013; 8:e76004. [PMID: 24146809 PMCID: PMC3797721 DOI: 10.1371/journal.pone.0076004] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 08/17/2013] [Indexed: 12/20/2022] Open
Abstract
Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
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Affiliation(s)
- Shuji Takada
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
- * E-mail: (ST); (HA)
| | - Tempei Sato
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yoshiaki Ito
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Satoshi Yamashita
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Tomoko Kato
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Miyuri Kawasumi
- Center for Experimental Animal, Tokyo Medical and Dental University, Tokyo, Japan
| | - Masami Kanai-Azuma
- Center for Experimental Animal, Tokyo Medical and Dental University, Tokyo, Japan
| | - Arisa Igarashi
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Tomomi Kato
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Moe Tamano
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
| | - Hiroshi Asahara
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
- CREST, Japan Science and Technology Agency (JST), Saitama, Japan
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America
- * E-mail: (ST); (HA)
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Menke DB. Engineering subtle targeted mutations into the mouse genome. Genesis 2013; 51:605-18. [PMID: 23913666 DOI: 10.1002/dvg.22422] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Revised: 07/25/2013] [Accepted: 07/26/2013] [Indexed: 12/13/2022]
Abstract
Homologous recombination in embryonic stem (ES) cells offers an exquisitely precise mechanism to introduce targeted modifications to the mouse genome. This ability to produce specific alterations to the mouse genome has become an essential tool for the analysis of gene function and the development of mouse models of human disease. Of the many thousands of mouse alleles that have been generated by gene targeting, the majority are designed to completely ablate gene function, to create conditional alleles that are inactivated in the presence of Cre recombinase, or to produce reporter alleles that label-specific tissues or cell populations (Eppig et al., 2012, Nucleic Acids Res 40:D881-D886). However, there is a variety of powerful motivations for the introduction of subtle targeted mutations (STMs) such as point mutations, small deletions, or small insertions into the mouse genome. The introduction of STMs allows the ablation of specific transcript isoforms, permits the functional investigation of particular domains or amino acids within a protein, provides the ability to study the role of specific sites with in cis-regulatory elements, and can result in better mouse models of human genetic disorders. In this review, I examine the current strategies that are commonly used to introduce STMs into the mouse genome and highlight new gene targeting technologies, including TALENs and CRISPR/Cas, which are likely to influence the future of gene targeting in mice.
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Affiliation(s)
- Douglas B Menke
- Department of Genetics, University of Georgia, Athens, Georgia
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Abstract
Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.
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