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Sahin GN, Seli E. Gene editing using CRISPR-Cas9 technology: potential implications in assisted reproduction. Curr Opin Obstet Gynecol 2025; 37:141-148. [PMID: 40232991 DOI: 10.1097/gco.0000000000001022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
PURPOSE OF REVIEW This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings. RECENT FINDINGS CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms. SUMMARY CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.
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Affiliation(s)
- Gizem Nur Sahin
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, Connecticut, USA
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2
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Sánchez-Gómez C, Posé D, Martín-Pizarro C. Genome Editing by CRISPR/Cas9 in Polyploids. Methods Mol Biol 2023; 2545:459-473. [PMID: 36720828 DOI: 10.1007/978-1-0716-2561-3_24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
CRISPR/Cas system has been widely used for genome editing in the past few years. Even though it has been performed in many polyploid species to date, its efficient accomplishment in these organisms is still a challenge. The presence of multiple homoeologous genes as targets for their editing requires more rigorous work and specific needs to assess successful genome editing. Here, we describe a general stepwise protocol to select target sites, design sgRNAs, indicate vector requirements, and screen CRISPR/Cas9-mediated genome editing in polyploid species.
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Affiliation(s)
- Carlos Sánchez-Gómez
- Departamento de Mejora Genética y Biotecnología, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM), Universidad de Málaga - Consejo Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, UMA, Málaga, Spain
| | - David Posé
- Departamento de Mejora Genética y Biotecnología, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM), Universidad de Málaga - Consejo Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, UMA, Málaga, Spain
| | - Carmen Martín-Pizarro
- Departamento de Mejora Genética y Biotecnología, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM), Universidad de Málaga - Consejo Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, UMA, Málaga, Spain.
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3
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Xie D, Deng T, Zhai Z, Sun T, Xu Y. The cellular model for Alzheimer's disease research: PC12 cells. Front Mol Neurosci 2023; 15:1016559. [PMID: 36683856 PMCID: PMC9846650 DOI: 10.3389/fnmol.2022.1016559] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2022] [Accepted: 12/08/2022] [Indexed: 01/06/2023] Open
Abstract
Alzheimer's disease (AD) is a common age-related neurodegenerative disease characterized by progressive cognitive decline and irreversible memory impairment. Currently, several studies have failed to fully elucidate AD's cellular and molecular mechanisms. For this purpose, research on related cellular models may propose potential predictive models for the drug development of AD. Therefore, many cells characterized by neuronal properties are widely used to mimic the pathological process of AD, such as PC12, SH-SY5Y, and N2a, especially the PC12 pheochromocytoma cell line. Thus, this review covers the most systematic essay that used PC12 cells to study AD. We depict the cellular source, culture condition, differentiation methods, transfection methods, drugs inducing AD, general approaches (evaluation methods and metrics), and in vitro cellular models used in parallel with PC12 cells.
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Affiliation(s)
- Danni Xie
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Ting Deng
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Zhenwei Zhai
- School of Medical Information Engineering, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Tao Sun
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
- School of Medical Information Engineering, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Ying Xu
- TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China
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Uncovering a Phenomenon of Active Hormone Transcriptional Regulation during Early Somatic Embryogenesis in Medicago sativa. Int J Mol Sci 2022; 23:ijms23158633. [PMID: 35955760 PMCID: PMC9368939 DOI: 10.3390/ijms23158633] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 07/30/2022] [Accepted: 08/01/2022] [Indexed: 12/04/2022] Open
Abstract
Somatic embryogenesis (SE) is a developmental process in which somatic cells undergo dedifferentiation to become plant stem cells, and redifferentiation to become a whole embryo. SE is a prerequisite for molecular breeding and is an excellent platform to study cell development in the majority of plant species. However, the molecular mechanism involved in M. sativa somatic embryonic induction, embryonic and maturation is unclear. This study was designed to examine the differentially expressed genes (DEGs) and miRNA roles during somatic embryonic induction, embryonic and maturation. The cut cotyledon (ICE), non-embryogenic callus (NEC), embryogenic callus (EC) and cotyledon embryo (CE) were selected for transcriptome and small RNA sequencing. The results showed that 17,251 DEGs, and 177 known and 110 novel miRNAs families were involved in embryonic induction (ICE to NEC), embryonic (NEC to EC), and maturation (EC to CE). Expression patterns and functional classification analysis showed several novel genes and miRNAs involved in SE. Moreover, embryonic induction is an active process of molecular regulation, and hormonal signal transduction related to pathways involved in the whole SE. Finally, a miRNA–target interaction network was proposed during M. sativa SE. This study provides novel perspectives to comprehend the molecular mechanisms in M. sativa SE.
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Mennati A, Rostamizadeh K, Manjili HK, Fathi M, Danafar H. Co-delivery of siRNA and lycopene encapsulated hybrid lipid nanoparticles for dual silencing of insulin-like growth factor 1 receptor in MCF-7 breast cancer cell line. Int J Biol Macromol 2022; 200:335-349. [PMID: 34999039 DOI: 10.1016/j.ijbiomac.2021.12.197] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 12/29/2021] [Accepted: 12/31/2021] [Indexed: 12/12/2022]
Abstract
Insulin-like growth factor-1 receptor (IGF-1R) is expressed in malignant and normal breast tissue, and its intermittent activation by multiple IGF-1 signaling pathways leads to neoplasm cell proliferation, impaired apoptosis, increased survival, and resistance to cytotoxic therapeutic agents. Therefore, simultaneous suppression of the receptor and its cognate ligand would be a powerful promising strategy inhibiting malignant phenotypes of breast cancer cells. In the present study, Methoxypoly(ethylene glycol) - Poly(caprolactone) was hybridized with Dimethyldioctadecylammonium bromide (DDAB) cationic lipid (mPEG-PCL-DDAB) nanoparticles (NPs) and used as a carrier for simultaneous delivery of lycopene and insulin-like growth factor 1 receptor-specific lycopene encapsulated-mPEG-PCL-DDAB nanoparticle/siRNA to MCF-7 breast cancer cells. Then, the antitumor effects of this construct were evaluated in vitro. The results demonstrated that the synthesized mPEG-PCL-DDAB nanoparticle had suitable physicochemical properties. The use of mPEG-PCL-DDAB nanoparticle-loaded anti-insulin-like growth factor 1 receptor-siRNA and lycopene dramatically induced the process of apoptosis and arrested cell cycle in the MCF-7 tumor cell lines. In general, the findings of this study demonstrated the potency of mPEG-PCL-DDAB nanoparticles for dual delivery of siRNA, and lycopene in breast cancer cell lines followed the induction of apoptosis.
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Affiliation(s)
- Afsaneh Mennati
- Zanjan Pharmaceutical Nanotechnology Research Center, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran; Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran; Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Kobra Rostamizadeh
- Zanjan Pharmaceutical Nanotechnology Research Center, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Hamidreza Kheiri Manjili
- Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
| | - Mojtaba Fathi
- Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran; Department of Biochemistry and Genetics, Qazvin University of Medical Sciences, Qazvin, Iran.
| | - Hossein Danafar
- Zanjan Pharmaceutical Nanotechnology Research Center, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran; Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
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Irwin AB, Bahabry R, Lubin FD. A putative role for lncRNAs in epigenetic regulation of memory. Neurochem Int 2021; 150:105184. [PMID: 34530054 PMCID: PMC8552959 DOI: 10.1016/j.neuint.2021.105184] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 08/29/2021] [Accepted: 08/31/2021] [Indexed: 12/12/2022]
Abstract
The central dogma of molecular genetics is defined as encoded genetic information within DNA, transcribed into messenger RNA, which contain the instructions for protein synthesis, thus imparting cellular functionality and ultimately life. This molecular genetic theory has given birth to the field of neuroepigenetics, and it is now well established that epigenetic regulation of gene transcription is critical to the learning and memory process. In this review, we address a potential role for a relatively new player in the field of epigenetic crosstalk - long non-coding RNAs (lncRNAs). First, we briefly summarize epigenetic mechanisms in memory formation and examine what little is known about the emerging role of lncRNAs during this process. We then focus discussions on how lncRNAs interact with epigenetic mechanisms to control transcriptional programs under various conditions in the brain, and how this may be applied to regulation of gene expression necessary for memory formation. Next, we explore how epigenetic crosstalk in turn serves to regulate expression of various individual lncRNAs themselves. To highlight the importance of further exploring the role of lncRNA in epigenetic regulation of gene expression, we consider the significant relationship between lncRNA dysregulation and declining memory reserve with aging, Alzheimer's disease, and epilepsy, as well as the promise of novel therapeutic interventions. Finally, we conclude with a discussion of the critical questions that remain to be answered regarding a role for lncRNA in memory.
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Affiliation(s)
- Ashleigh B Irwin
- Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Rudhab Bahabry
- Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Farah D Lubin
- Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
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Li Y, Feng H, Jin L, Xin X, Yuan Q. A novel vector-based RNAi method using mouse U6 promoter-driven shRNA expression in the filamentous fungus Blakeslea trispora. Biotechnol Lett 2021; 43:1821-1830. [PMID: 34185215 DOI: 10.1007/s10529-021-03155-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Accepted: 06/10/2021] [Indexed: 11/24/2022]
Abstract
PURPOSE There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi. METHODS Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora. RESULTS We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi. CONCLUSIONS The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.
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Affiliation(s)
- Ye Li
- Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China
| | - Hui Feng
- Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China
| | - Lihua Jin
- Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China
| | - Xiulan Xin
- Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China
| | - Qipeng Yuan
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China.
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Araújo RS, Bitoque DB, Silva GA. Development of strategies to modulate gene expression of angiogenesis-related molecules in the retina. Gene 2021; 791:145724. [PMID: 34010703 DOI: 10.1016/j.gene.2021.145724] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Revised: 05/10/2021] [Accepted: 05/13/2021] [Indexed: 12/14/2022]
Abstract
Intravitreal anti-vascular endothelial growth factor agents are the gold standard treatment of ocular neovascular diseases. However, their short-term efficacy implies frequent intravitreal injections. Gene therapy has the ability to provide longer duration of the therapeutic effect. We have previously described the effectiveness of the self-replicating episomal vector, pEPito, in long-term gene expression in mouse retina. In this study, we evaluated different constructs to overexpress pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor, and simultaneously, to silence placental growth factor (PlGF), a key player in neovascularization. We employed the human cytomegalovirus promoter to drive the expression of PEDF and PlGF shRNA, in conjunction with cis-acting ribozymes, using pEPito as expressing vector. Our results demonstrated that the non-viral systems were able to efficiently promote a sustained increase of the PEDF: PlGF ratio in the mice retina, decreased in pathological conditions. This innovative approach could open avenues for the development of new therapeutic strategies.
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Affiliation(s)
- Rute S Araújo
- iNOVA4Health, CEDOC, NOVA Medical School, Universidade Nova de, Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal; Bioengineering - Cell Therapies and Regenerative Medicine PhD Program, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
| | - Diogo B Bitoque
- iNOVA4Health, CEDOC, NOVA Medical School, Universidade Nova de, Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal
| | - Gabriela A Silva
- iNOVA4Health, CEDOC, NOVA Medical School, Universidade Nova de, Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal; NOVA Medical School, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal.
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MicroRNAs Regulating Autophagy in Neurodegeneration. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1208:191-264. [PMID: 34260028 DOI: 10.1007/978-981-16-2830-6_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Social and economic impacts of neurodegenerative diseases (NDs) become more prominent in our constantly aging population. Currently, due to the lack of knowledge about the aetiology of most NDs, only symptomatic treatment is available for patients. Hence, researchers and clinicians are in need of solid studies on pathological mechanisms of NDs. Autophagy promotes degradation of pathogenic proteins in NDs, while microRNAs post-transcriptionally regulate multiple signalling networks including autophagy. This chapter will critically discuss current research advancements in the area of microRNAs regulating autophagy in NDs. Moreover, we will introduce basic strategies and techniques used in microRNA research. Delineation of the mechanisms contributing to NDs will result in development of better approaches for their early diagnosis and effective treatment.
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Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer. Cancers (Basel) 2020; 12:cancers12123695. [PMID: 33317042 PMCID: PMC7763270 DOI: 10.3390/cancers12123695] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 12/06/2020] [Accepted: 12/07/2020] [Indexed: 12/16/2022] Open
Abstract
Simple Summary Long non-coding RNAs (lncRNAs) are a recently discovered class of molecules in the cell, with potential to be utilized as therapeutic targets in cancer. A number of lncRNAs have been described to play important roles in tumor progression and drive molecular processes involved in cell proliferation, apoptosis or invasion. However, the vast majority of lncRNAs have not been studied in the context of cancer thus far. With the advent of CRISPR/Cas genome editing, high-throughput functional screening approaches to identify lncRNAs that impact cancer growth are becoming more accessible. Here, we review currently available methods to study hundreds to thousands of lncRNAs in parallel to elucidate their role in tumorigenesis and cancer progression. Abstract Recent technological advancements such as CRISPR/Cas-based systems enable multiplexed, high-throughput screening for new therapeutic targets in cancer. While numerous functional screens have been performed on protein-coding genes to date, long non-coding RNAs (lncRNAs) represent an emerging class of potential oncogenes and tumor suppressors, with only a handful of large-scale screens performed thus far. Here, we review in detail currently available screening approaches to identify new lncRNA drivers of tumorigenesis and tumor progression. We discuss the various approaches of genomic and transcriptional targeting using CRISPR/Cas9, as well as methods to post-transcriptionally target lncRNAs via RNA interference (RNAi), antisense oligonucleotides (ASOs) and CRISPR/Cas13. We discuss potential advantages, caveats and future applications of each method to provide an overview and guide on investigating lncRNAs as new therapeutic targets in cancer.
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Kumar S, Gonzalez EA, Rameshwar P, Etchegaray JP. Non-Coding RNAs as Mediators of Epigenetic Changes in Malignancies. Cancers (Basel) 2020; 12:E3657. [PMID: 33291485 PMCID: PMC7762117 DOI: 10.3390/cancers12123657] [Citation(s) in RCA: 74] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 12/01/2020] [Accepted: 12/03/2020] [Indexed: 12/12/2022] Open
Abstract
Non-coding RNAs (ncRNAs) are untranslated RNA molecules that regulate gene expressions. NcRNAs include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), circular RNAs (cRNAs) and piwi-interacting RNAs (piRNAs). This review focuses on two types of ncRNAs: microRNAs (miRNAs) or short interfering RNAs (siRNAs) and long non-coding RNAs (lncRNAs). We highlight the mechanisms by which miRNAs and lncRNAs impact the epigenome in the context of cancer. Both miRNAs and lncRNAs have the ability to interact with numerous epigenetic modifiers and transcription factors to influence gene expression. The aberrant expression of these ncRNAs is associated with the development and progression of tumors. The primary reason for their deregulated expression can be attributed to epigenetic alterations. Epigenetic alterations can cause the misregulation of ncRNAs. The experimental evidence indicated that most abnormally expressed ncRNAs impact cellular proliferation and apoptotic pathways, and such changes are cancer-dependent. In vitro and in vivo experiments show that, depending on the cancer type, either the upregulation or downregulation of ncRNAs can prevent the proliferation and progression of cancer. Therefore, a better understanding on how ncRNAs impact tumorigenesis could serve to develop new therapeutic treatments. Here, we review the involvement of ncRNAs in cancer epigenetics and highlight their use in clinical therapy.
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Affiliation(s)
- Subhasree Kumar
- Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA; (S.K.); (E.A.G.)
| | - Edward A. Gonzalez
- Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA; (S.K.); (E.A.G.)
| | - Pranela Rameshwar
- Department of Medicine, Hematology/Oncology, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ 07103, USA
| | - Jean-Pierre Etchegaray
- Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA; (S.K.); (E.A.G.)
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Ando H, Ishida T. An RNAi therapeutic, DFP-10825, for intraperitoneal and intrapleural malignant cancers. Adv Drug Deliv Rev 2020; 154-155:27-36. [PMID: 32781056 DOI: 10.1016/j.addr.2020.08.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 07/31/2020] [Accepted: 08/06/2020] [Indexed: 12/16/2022]
Abstract
RNA interference (RNAi), a potent post-transcriptional gene-silencing action, has received considerable attentions as a novel therapeutic tool to treat intractable cancers. In recent days, we have developed a novel RNAi-based therapeutic formulation, DFP-10825, for the treatment of intractable advanced cancers developed in coelomic cavities. DFP-10825 was composed of chemically synthesized short hairpin RNA (shRNA) against thymidylate synthase (TS), a key enzyme for cancer proliferation, and cationic liposomes, and achieved high therapeutic effect on the mouse models of peritoneally disseminated gastric and ovarian cancers and malignant pleural mesothelioma without severe side effects by intracoelomic direct treatment. We further designed a freeze-dried DFP-10825 formulation for mass industrial production. DFP-10825 is undergoing in pre-clinical phase and goes to clinical trials. This review introduces a DFP-10825 formulation, a potent novel RNAi-based therapeutic maximizing the benefit of RNAi molecule (shRNA).
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Wu Y, Song T, Chen P, Jiang X, Wang Q, Chen Q. Prolonged siRNA expression in mammalian cells using an Epstein-Barr virus-based plasmid expression system. Biochem Biophys Res Commun 2020; 529:51-56. [PMID: 32560818 DOI: 10.1016/j.bbrc.2020.05.219] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2020] [Accepted: 05/31/2020] [Indexed: 11/19/2022]
Abstract
RNA interference (RNAi) is a powerful tool in gene function analysis and disease treatment, especially diseases that are 'undruggable' by classical small molecules. However, the RNAi applications are limited due to some defects, such as short duration and toxic side effects. New strategies are still needed to improve RNAi applications. Previous studies have illustrated that Epstein-Barr virus nuclear antigen 1 (EBNA-1) and the origin of plasmid replication (oriP) are critical factors for EBV latent gene expression, which can keep the replication of the EBV genome as an extrachromosomal element for a relatively long time. Here we report a plasmid expression system on the base of oriP and EBNA-1, which could produce protein as well as short interfering RNAs(siRNAs) for a long time in mammalian cells. siRNA expression mediated by this system causes efficient and specific down-regulation of gene expression. Except for analyzing gene function, this study also provided a new optional and practical way for protein and/or RNAi-based therapies that require enduring effect.
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Affiliation(s)
- Yan Wu
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China
| | - Tianqiang Song
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China
| | - Peipei Chen
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China
| | - Xiaohong Jiang
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China
| | - Qiang Wang
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China
| | - Qihan Chen
- State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, People's Republic of China.
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Cytoplasmic expression of EGFR shRNA using a modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR silencing and prolongs antitumor effects. Biochem Pharmacol 2020; 171:113735. [DOI: 10.1016/j.bcp.2019.113735] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Accepted: 11/26/2019] [Indexed: 12/18/2022]
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15
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Song R, Zhai Q, Sun L, Huang E, Zhang Y, Zhu Y, Guo Q, Tian Y, Zhao B, Lu H. CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective. Appl Microbiol Biotechnol 2019; 103:6919-6932. [PMID: 31332488 PMCID: PMC6690858 DOI: 10.1007/s00253-019-10007-w] [Citation(s) in RCA: 98] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2019] [Revised: 06/28/2019] [Accepted: 07/01/2019] [Indexed: 12/16/2022]
Abstract
Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.
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Affiliation(s)
- Runjie Song
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Qing Zhai
- Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa, 850000, Tibet, China
| | - Lu Sun
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Enxia Huang
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Yu Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Yanli Zhu
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Qingyun Guo
- Qinghai Academy of Agriculture and Forestry Sciences, Qinghai University/Key Laboratory of Agricultural Integrated Pest Management, Qinghai Province/State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining, 810016, Qinghai, China.
| | - Yanan Tian
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX, 77843, USA
| | - Baoyu Zhao
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China
| | - Hao Lu
- College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
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16
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Abstract
Small silencing RNAs have provided powerful reverse genetics tools and have opened new areas of research. This introduction describes the use of RNAi to suppress expression of individual genes for loss-of-function analysis. It also summarizes methods for measuring specific and global changes in small RNA expression, as well as methods to inhibit the function of individual endogenous small RNA species such as miRNAs.
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17
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Kwak SY, Han HD, Ahn HJ. A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry. Sci Rep 2019; 9:2993. [PMID: 30816180 PMCID: PMC6395690 DOI: 10.1038/s41598-019-39407-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Accepted: 01/24/2019] [Indexed: 12/30/2022] Open
Abstract
The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.
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Affiliation(s)
- Seo Young Kwak
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, South Korea
| | - Hee Dong Han
- Department of Immunology, School of Medicine, Konkuk University, Chungju, South Korea
| | - Hyung Jun Ahn
- Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, South Korea.
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18
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Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation. Biotechnol Adv 2019; 37:107361. [PMID: 30825514 DOI: 10.1016/j.biotechadv.2019.02.017] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 02/11/2019] [Accepted: 02/23/2019] [Indexed: 12/26/2022]
Abstract
Fungal strain engineering is commonly used in many areas of biotechnology, including the production of plant biomass degrading enzymes. Its aim varies from the production of specific enzymes to overall increased enzyme production levels and modification of the composition of the enzyme set that is produced by the fungus. Strain engineering involves a diverse range of methodologies, including classical mutagenesis, genetic engineering and genome editing. In this review, the main approaches for strain engineering of filamentous fungi in the field of plant biomass degradation will be discussed, including recent and not yet implemented methods, such as CRISPR/Cas9 genome editing and adaptive evolution.
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19
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Grunwald HA, Gantz VM, Poplawski G, Xu XRS, Bier E, Cooper KL. Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline. Nature 2019; 566:105-109. [PMID: 30675057 PMCID: PMC6367021 DOI: 10.1038/s41586-019-0875-2] [Citation(s) in RCA: 139] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2018] [Accepted: 12/07/2018] [Indexed: 01/02/2023]
Abstract
A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
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Affiliation(s)
- Hannah A Grunwald
- Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA
| | - Valentino M Gantz
- Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA
| | - Gunnar Poplawski
- Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA
- Department of Medicine, National University of Singapore, Singapore, Singapore
| | - Xiang-Ru S Xu
- Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA
| | - Ethan Bier
- Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA
- Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA
| | - Kimberly L Cooper
- Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.
- Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA.
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20
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Hong Z, Mao X, You J, Liu Z, Shi Y. An Evaluation of HER2-Positive Ovarian Carcinoma Xenografts: From a Novel Therapy to a Noninvasive Monitoring Method. Cancer Biother Radiopharm 2018; 33:411-419. [PMID: 30052070 DOI: 10.1089/cbr.2018.2516] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Affiliation(s)
- Zhihui Hong
- Department of Nuclear Medicine, The Second Affiliated Hospital of Soochow University, Suzhou City, People's Republic of China
| | - Xinping Mao
- Division of Medical Imageology, GanSu University of Chinese Medicine, Lanzhou City, People's Republic of China
| | - Jiaxi You
- Department of Nuclear Medicine, The Second Affiliated Hospital of Soochow University, Suzhou City, People's Republic of China
| | - Zengli Liu
- Department of Nuclear Medicine, The Second Affiliated Hospital of Soochow University, Suzhou City, People's Republic of China
| | - Yizhen Shi
- Department of Nuclear Medicine, The Second Affiliated Hospital of Soochow University, Suzhou City, People's Republic of China
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21
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Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3'-overhang. Nat Commun 2018; 9:3651. [PMID: 30194297 PMCID: PMC6128929 DOI: 10.1038/s41467-018-06129-w] [Citation(s) in RCA: 117] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Accepted: 08/14/2018] [Indexed: 12/18/2022] Open
Abstract
Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U4AU4 3'-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.
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22
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Zhao TP, Wang XL, Han YM. Knockdown of p57 gene inhibits breast cancer cell proliferation. Oncol Lett 2018; 16:55-58. [PMID: 29928386 PMCID: PMC6006381 DOI: 10.3892/ol.2018.8605] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 11/14/2016] [Indexed: 12/02/2022] Open
Abstract
The aim of the study was to investigate possible effects of p57 on the growth of the human MCF-7 and rat SHZ-88 breast cancer cell lines. Specific oligonucleotide sequences containing small hairpin structure were inserted into a small interfering RNA (siRNA) expression vector. The human MCF-7 and rat SHZ-88 breast cancer cell lines were transfected with recombinant plasmids. The p57 gene expression was blocked in the human MCF-7 breast and rat SHZ-88 breast cancer cells, using chemically modified siRNA. The p57 expression level was evaluated using quantitative polymerase chain reaction (qPCR) and western blot analysis. Immunofluorescence was conducted to detect p57 expression in the breast cancer cells. Tetrazolium blue (MTT) method was employed to detect the effect of p57 inhibition on the proliferation of the MCF-7 and SHZ-88 cell lines. Cell proliferation in the experimental group was significantly reduced. Immunofluorescence assay results showed p57 siRNA effectively inhibited the p57 level in the MCF-7 and SHZ-88 cells. RT-PCR results showed that 48 h after transfection, the p57 mRNA level in the transfected group was significantly lower compared with the control group. In conclusion, p57 effectively inhibited the proliferation of breast cancer after stable interference.
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Affiliation(s)
- Tai Ping Zhao
- College of Health Sciences, Guangzhou Medical University, Guangzhou, Guangdong 510450, P.R. China
| | - Xin Liang Wang
- Guangzhou First People's Hospital, Guangzhou, Guangdong 510180, P.R. China
| | - Yi Min Han
- Department of Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang 150081, P.R. China
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23
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Williams RM, Senanayake U, Artibani M, Taylor G, Wells D, Ahmed AA, Sauka-Spengler T. Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo. Development 2018; 145:dev.160333. [PMID: 29386245 DOI: 10.1242/dev.160333] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2017] [Accepted: 01/21/2018] [Indexed: 12/17/2022]
Abstract
CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.
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Affiliation(s)
- Ruth M Williams
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
| | - Upeka Senanayake
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
| | - Mara Artibani
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK.,University of Oxford, Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, UK
| | - Gunes Taylor
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
| | - Daniel Wells
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
| | - Ahmed Ashour Ahmed
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK.,University of Oxford, Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, UK.,Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women's Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK
| | - Tatjana Sauka-Spengler
- University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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24
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Abstract
CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.
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Affiliation(s)
- Neena K. Pyzocha
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Sidi Chen
- Department of Genetics, Yale University, 333 Cedar Street, New Haven, Connecticut 06510, United States
- System Biology Institute, 850 West Campus Drive, ISTC 361, West Haven, Connecticut 06516, United States
- MCGD Program, Yale University, 333 Cedar Street, New Haven, Connecticut 06510, United States
- Immunobiology Program, Yale University, 300 Cedar Street, New Haven, Connecticut 06520, United States
- Comprehensive Cancer Center, Yale University, New Haven, Connecticut 06510, United States
- Stem Cell Center, Yale University, New Haven, Connecticut 06510, United States
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25
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Xiao H, Yang R, Yang F, Zhao Y, Liu Y. Construction of a plasmid vector containing epidermal growth factor receptor and C-Jun shRNA. Arch Dermatol Res 2018; 310:241-243. [PMID: 29353331 DOI: 10.1007/s00403-017-1803-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2017] [Revised: 12/13/2017] [Accepted: 12/19/2017] [Indexed: 11/28/2022]
Abstract
The objective of this study was to construct a plasmid vector for EGFR-hm-1 and C-Junh-825 small interfering RNA (siRNA). EGFR-hm-1 and C-Jun-hm-825 oligonucleotide fragments were synthesized and short hairpin RNA (shRNA) were amplified by PCR. Plasmids were isolated from E. coli TOP10 bacterium by restriction enzyme digestion using pst1 and BamH1 and oligonucleotide fragments were cloned into the pSilencer plasmid containing the U6 promoter. Recombinant clones were generated by transforming JM109 competent cells with plasmid vectors containing shRNA molecules. 58 base-paired EGFR-hm-1 and 59 base-paired C-Jun-hm-825 oligonucleotide fragments were isolated. The fragments were 100% homologous with human sequences available on GenBank. The recombinant pSilencer1.0 vector containing a 58-bp EGFR-hm-1 and 59-bp C-Jun-hm-825 fragment was constructed. These vectors have the potential to be used as treatment to combat skin photoaging under UV exposure.
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Affiliation(s)
- Hong Xiao
- Department of Plastic Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming, 650103, Yunnan, China
| | - Ruinian Yang
- Department of Plastic Surgery, YESTAR Aesthetic Plastic Hospital, Kunming, Yunnan, China
| | - Fang Yang
- Department of Plastic Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming, 650103, Yunnan, China
| | - Yanan Zhao
- Department of Plastic Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming, 650103, Yunnan, China
| | - Yin Liu
- Department of Plastic Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming, 650103, Yunnan, China.
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26
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Computational design of small interfering RNAs and small hairpin RNAs to silence mutated P53 gene expressions. INFORMATICS IN MEDICINE UNLOCKED 2018. [DOI: 10.1016/j.imu.2018.04.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
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27
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Abstract
Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude for a human body for etiological and pathological research of diseases. All pharmaceutically developed medicines must be proven to be safe and effective in animals before approval by the Food and Drug Administration (FDA) to be used in clinical trials. To this end, the transgenic animal models of diseases serve as the front line of drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA)-related research. MiRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts (mRNAs) that contain either complete or partial complementarity to the miRNA, are useful for the design of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis spp.), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By the insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerases type II (Pol-II), coexpressed with the encoding gene transcripts, and excised out of the encoding gene transcripts by intracellular RNA splicing and processing mechanisms. In conjunction with retroviral transfection, the designed hairpin-like pre-miRNA construct has also been placed in the intron regions of a cellular gene for tissue-specific expression, specifically regulated by the gene promoter of interest. Because the retroviral vectors are integrated into the genome of its host cells, we can select and propagate the most effective transgenic animals to form a stable model line for further research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA expression system reported previously, which has been proven to be useful for studying miRNA function as well as the related gene regulation in vivo.
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Affiliation(s)
- Shi-Lung Lin
- Division of Regenerative Medicine, WJWU & LYNN Institute for Stem Cell Research, Santa Fe Springs, CA, USA.
| | - Shin-Ju E Chang
- Division of Regenerative Medicine, WJWU & LYNN Institute for Stem Cell Research, Santa Fe Springs, CA, USA
| | - Shao-Yao Ying
- Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
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28
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Shimai K, Kusakabe TG. The Use of cis-Regulatory DNAs as Molecular Tools. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018. [DOI: 10.1007/978-981-10-7545-2_6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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29
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Long L, Guo DD, Gao W, Yang WW, Hou LP, Ma XN, Miao YC, Botella JR, Song CP. Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression. PLANT METHODS 2018; 14:85. [PMID: 30305839 PMCID: PMC6169012 DOI: 10.1186/s13007-018-0353-0] [Citation(s) in RCA: 62] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Accepted: 09/26/2018] [Indexed: 05/20/2023]
Abstract
BACKGROUND When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency. RESULTS In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6-29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the AtU6-29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times. CONCLUSIONS This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.
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Affiliation(s)
- Lu Long
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Dan-Dan Guo
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Wei Gao
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Wen-Wen Yang
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Li-Pan Hou
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Xiao-Nan Ma
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Yu-Chen Miao
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
| | - Jose Ramon Botella
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
- School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072 Australia
| | - Chun-Peng Song
- State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People’s Republic of China
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30
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Gao Z, Herrera-Carrillo E, Berkhout B. Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III. MOLECULAR THERAPY-NUCLEIC ACIDS 2017; 10:36-44. [PMID: 29499947 PMCID: PMC5725217 DOI: 10.1016/j.omtn.2017.11.006] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Revised: 11/15/2017] [Accepted: 11/15/2017] [Indexed: 12/29/2022]
Abstract
Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1). Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of ≥6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1–6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III.
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Affiliation(s)
- Zongliang Gao
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Elena Herrera-Carrillo
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Ben Berkhout
- Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
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31
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Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells. Methods Mol Biol 2017. [PMID: 28674803 DOI: 10.1007/978-1-4939-7108-4_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
The development of the CRISPR/Cas9 technology has provided powerful methods to target genetic alterations. However, investigating the function of genes essential for cell survival remains problematic, because genetic ablation of these genes results in cell death. As a consequence, cells recombined at the targeted gene and fully depleted of the gene product cannot be obtained. RNA interference is well suited for the study of essential genes, but this approach often results in a partial depletion of the targeted gene product, which can lead to misinterpretations. We previously developed the pHYPER shRNA vector, a high efficiency RNA interference vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We provide here a pHYPER-based protocol optimized to study the function of essential gene products in mouse embryonic stem cells.
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32
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Abstract
As eukaryotes, filamentous fungi share many features with humans, and they produce numerous active metabolites, some of which are toxic. Traditional genetic approaches are generally inefficient, but the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system that has been widely used for basic research on bacteria, mammals and plants offers a simple, fast, versatile technology for systemic research on filamentous fungi. In this review, we summarized the current knowledge on Cas9 and its variants, various selective markers used to screen positive clones, different ways used to detect off-target mutations, and different approaches used to express and transform the CRISPR complex. We also highlight several methods that improve the nuclease specificity and efficiency, and discuss current and potential applications of CRISPR/Cas9 system in filamentous fungi for pathogenesis decoding, confirmation of the gene and pathway, bioenergy process, drug discovery, and chromatin dynamics. We also describe how the synthetic gene circuit of CRISPR/Cas9 systems has been used in the response to various complex environmental signals to redirect metabolite flux and ensure continuous metabolite biosynthesis.
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Affiliation(s)
- Huaxiang Deng
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China
| | - Ruijie Gao
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China
| | - Xiangru Liao
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China.
| | - Yujie Cai
- The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People's Republic of China.
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Mishra DK, Balekar N, Mishra PK. Nanoengineered strategies for siRNA delivery: from target assessment to cancer therapeutic efficacy. Drug Deliv Transl Res 2017; 7:346-358. [PMID: 28050890 DOI: 10.1007/s13346-016-0352-5] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
The promise of RNA interference (RNAi) technology in cancer therapeutics aims to deliver small interfering RNA (siRNA) for silencing of gene expression in cell type-specific pathway. However, the challenge for the delivery of stable siRNA is hindered by an immune-hostile tumor microenvironment and physiological barriers of the circulatory system. Therefore, the development and validation of safe, stable, and efficient nanoengineered delivery systems are highly essential for effective delivery of siRNA into cancer cells. This review focuses on gene-silencing mechanisms, challenges to siRNA delivery, design and delivery of nanocarrier systems, ongoing clinical trials, and translational prospects for siRNA-mediated cancer therapeutics.
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Affiliation(s)
| | - Neelam Balekar
- IPS Academy, College of Pharmacy, A. B. Road, Indore, MP, 452 012, India
| | - Pradyumna Kumar Mishra
- Department of Molecular Biology, National Institute for Research in Environmental Health, Indian Council of Medical Research (ICMR), Bhopal, India
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34
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Shali H, Shabani M, Pourgholi F, Hajivalili M, Aghebati-Maleki L, Jadidi-Niaragh F, Baradaran B, Movassaghpour Akbari AA, Younesi V, Yousefi M. Co-delivery of insulin-like growth factor 1 receptor specific siRNA and doxorubicin using chitosan-based nanoparticles enhanced anticancer efficacy in A549 lung cancer cell line. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2017; 46:293-302. [DOI: 10.1080/21691401.2017.1307212] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Affiliation(s)
- Hajar Shali
- Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
- Student’s Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mahdi Shabani
- Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Fatemeh Pourgholi
- Department of Immunology, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mahsa Hajivalili
- Department of Immunology, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Farhad Jadidi-Niaragh
- Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Behzad Baradaran
- Department of Immunology, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Vahid Younesi
- Pishtaz Teb Diagnostics, Tehran, Iran
- Faculty of Paramedical Sciences, Alborz University of Medical Sciences, Karaj, Iran
| | - Mehdi Yousefi
- Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
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35
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Wettengel J, Reautschnig P, Geisler S, Kahle PJ, Stafforst T. Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy. Nucleic Acids Res 2017; 45:2797-2808. [PMID: 27907896 PMCID: PMC5389476 DOI: 10.1093/nar/gkw911] [Citation(s) in RCA: 66] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2016] [Revised: 09/27/2016] [Accepted: 09/30/2016] [Indexed: 02/06/2023] Open
Abstract
Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.
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Affiliation(s)
- Jacqueline Wettengel
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
| | - Philipp Reautschnig
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
| | - Sven Geisler
- Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller-Strasse 27, 72076 Tübingen, Germany
- German Center for Neurodegenerative Diseases, Otfried-Müller-Strasse 23, 72076 Tübingen, Germany
| | - Philipp J. Kahle
- Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried-Müller-Strasse 27, 72076 Tübingen, Germany
- German Center for Neurodegenerative Diseases, Otfried-Müller-Strasse 23, 72076 Tübingen, Germany
| | - Thorsten Stafforst
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
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36
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Mathupala SP, Guthikonda M, Sloan AE. RNAi Based Approaches to the Treatment of Malignant Glioma. Technol Cancer Res Treat 2016; 5:261-9. [PMID: 16700622 DOI: 10.1177/153303460600500313] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
RNA interference (RNAi) is a recently discovered, powerful molecular mechanism that can be harnessed to engineer gene-specific silencing in mammalian tissues. A mechanism, where short double-stranded RNA (dsRNA) molecules, when introduced into cells elicit specific “knock-down” of gene expression via degradation of targeted messenger RNA, has lately become the technique of choice for analysis of gene function in oncology research. Thus, RNAi is currently being extensively evaluated as a potential therapeutic strategy against malignant gliomas, since surgical, radiological, and chemotherapeutic interventions during the past few decades have done little to improve the poor prognosis rate for patients with these dreaded tumors. This review summarizes the pre-clinical studies that are currently underway to test the validity of RNAi as a potential therapeutic strategy against malignant gliomas, and discusses the potential technical Hurdles that remain to be overcome before the technique can become a promising clinical therapy to combat this frequently lethal disease.
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Affiliation(s)
- Saroj P Mathupala
- Department of Neurological Surgery, Karmanos Cancer Institute, Wayne State University School of Medicine, 808 HWCRC, 4100 John R. Road, Detroit, MI 48201, USA.
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37
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Cai Y, Yi M, Chen D, Liu J, Guleng B, Ren J, Shi H. Trefoil factor family 2 expression inhibits gastric cancer cell growth and invasion in vitro via interactions with the transcription factor Sp3. Int J Mol Med 2016; 38:1474-1480. [PMID: 27668303 PMCID: PMC5065293 DOI: 10.3892/ijmm.2016.2739] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2015] [Accepted: 09/09/2016] [Indexed: 12/14/2022] Open
Abstract
The trefoil factor family (TFF) is a group of short secretory peptides of gastric mucous neck cells. The loss of TFF2 protein expression enhances gastric inflammation and occurs in gastric cancer. In this study, we examined the effect of TFF2 on gastric cancer cell lines in vitro and characterized the interaction between TFF2 and Sp3, including the mechanisms that mediate this interaction, using genomics and proteomics approaches, as well as genetics techniques, such as RNA interference and gene knockdown. Assays were performed to examine the role of TFF2 and Sp3 in cancer cell proliferation, invasion and migration. We found that TFF2 expression inhibited the proliferation and invasion capacity of gastric cancer cells, and induced apoptosis. TFF2 interacted with the Sp3 protein, as shown by immunofluorescence staining and immunoprecipitation with western blot analysis. Sp3 knockdown in gastric cancer cells antagonized TFF2 anti-tumor activity. Additionally, TFF2 upregulated the expression of pro-apoptotic proteins, such as Bid, but downregulated the expression of NF-κB and the anti-apoptotic proteins, Bcl-xL and Mcl-1. By contrast, Sp3 knockdown significantly blocked TFF2 activity, affecting the expression of these proteins. The data from our study demonstrate that the antitumor activity of TFF2 is mediated by an interaction with the Sp3 protein in gastric cancer cells. Additional in vivo and ex vivo warrned in order to fully characterize this interaction.
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Affiliation(s)
- Yiling Cai
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Mengting Yi
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Dajun Chen
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Jingjing Liu
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Bayasi Guleng
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Jianlin Ren
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
| | - Huaxiu Shi
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China
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38
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Shinohara ET, Lu B, Hallahan DE. The Use of Gene Therapy in Cancer Research and Treatment. Technol Cancer Res Treat 2016; 3:479-90. [PMID: 15453813 DOI: 10.1177/153303460400300509] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Gene therapy involves identifying a gene of interest and then manipulating the expression of this gene through a variety of techniques. Here we specifically address gene therapy's role in cancer research. This paper will encompass thoroughly investigated techniques such as cancer vaccines and suicide gene therapy and the latest advancements in and applications of these techniques. It will also cover newer techniques such as Antisense Oligonucleotides and small interfering RNAs and how these technologies are being developed and used. The use of gene therapy continues to expand in cancer research and has an integral role in the advancement of cancer treatment.
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Affiliation(s)
- E T Shinohara
- Department of Radiation Oncology, Vanderbilt University, 1301 22nd Avenue South, B-902, The Vanderbilt Clinic, Nashville, Tennessee 37232-5671, USA
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39
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Abstract
Short interfering RNAs (siRNAs) are as effective at targeting and silencing genes by RNA interference (RNAi) as long double-stranded RNAs (dsRNAs). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. siRNAs are also promising reagents for developing gene-specific therapeutics. Specifically, the inhibition of HIV-1 replication is particularly well-suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on recent advances in siRNA-based silencing technologies.
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Affiliation(s)
- Hiroshi Takaku
- Department of Life & Environmental Sciences and High Technology Research Center, Chiba Institute of Technology, Chiba, Japan.
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40
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Fakhr E, Zare F, Teimoori-Toolabi L. Precise and efficient siRNA design: a key point in competent gene silencing. Cancer Gene Ther 2016; 23:73-82. [DOI: 10.1038/cgt.2016.4] [Citation(s) in RCA: 126] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 02/01/2016] [Accepted: 02/01/2016] [Indexed: 12/14/2022]
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41
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Raitskin O, Patron NJ. Multi-gene engineering in plants with RNA-guided Cas9 nuclease. Curr Opin Biotechnol 2016; 37:69-75. [PMID: 26707469 DOI: 10.1016/j.copbio.2015.11.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2015] [Revised: 10/26/2015] [Accepted: 11/02/2015] [Indexed: 11/17/2022]
Abstract
The use of RNA-guided Cas9 endonuclease for the concurrent engineering of multiple genes has been demonstrated in a number of plant species. Although Cas9 is a large monomeric protein, the single guide RNA (sgRNA) that directs it to a specific DNA target sequence is small and easy to reprogram. It is therefore relatively simple to produce numerous sgRNAs to target multiple endogenous sequences. Several approaches to express multiple sgRNAs and Cas9 in plants for the purpose of simultaneous editing or transcriptional regulation of many genes have recently been reported.
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Affiliation(s)
- Oleg Raitskin
- The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK
| | - Nicola J Patron
- The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK.
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42
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Shao SL, Cui TT, Zhao W, Zhang WW, Xie ZL, Wang CH, Jia HS, Liu Q. RNAi-based knockdown of multidrug resistance-associated protein 1 is sufficient to reverse multidrug resistance of human lung cells. Asian Pac J Cancer Prev 2015; 15:10597-601. [PMID: 25605145 DOI: 10.7314/apjcp.2014.15.24.10597] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Up-regulation of multidrug resistance-associated protein 1 (MRP1) is regarded as one of the main causes for multidrug resistance (MDR) of tumor cells, leading to failure of chemotherapy-based treatment for a multitude of cancers. However, whether silencing the overexpressed MRP1 is sufficient to reverse MDR has yet to be validated. This study demonstrated that RNAi-based knockdown of MRP1 reversed the increased efflux ability and MDR efficiently. Two different short haipin RNAs (shRNAs) targeting MRP1 were designed and inserted into pSilence- 2.1-neo. The shRNA recombinant plasmids were transfected into cis-dichlorodiamineplatinum-resistant A549 lung (A549/DDP) cells, and then shRNA expressing cell clones were collected and maintained. Real time PCR and immunofluorescence staining for MRP1 revealed a high silent efficiency of these two shRNAs. Functionally, shRNA-expressing cells showed increased rhodamine 123 retention in A549/DDP cells, indicating reduced efflux ability of tumor cells in the absence of MRP1. Consistently, MRP1-silent cells exhibited decreased resistance to 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DDP, suggesting reversal of MDR in these tumor cells. Specifically, MRP1 knockdown increased the DDP-induced apoptosis of A549/DDP cells by increased trapping of their cell cycling in the G2 stage. Taken together, this study demonstrated that RNAi- based silencing of MRP1 is sufficient to reverse MDR in tumor cells, shedding light on possible novel clinical treatment of cancers.
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Affiliation(s)
- Shu-Li Shao
- College of Life Sciences and Agriculture and Forestry, Qiqihar University, Qiqihar, China E-mail :
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43
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Yan M, Wen J, Liang M, Lu Y, Kamata M, Chen ISY. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs. PLoS One 2015; 10:e0127986. [PMID: 26035832 PMCID: PMC4452785 DOI: 10.1371/journal.pone.0127986] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 04/21/2015] [Indexed: 01/25/2023] Open
Abstract
Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.
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Affiliation(s)
- Ming Yan
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America
- California NanoSystems Institute (CNSI), University of California Los Angeles, Los Angeles, California, United States of America
- University of California Los Angeles AIDS Institute, Los Angeles, California, United States of America
| | - Jing Wen
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America
- University of California Los Angeles AIDS Institute, Los Angeles, California, United States of America
| | - Min Liang
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America
| | - Yunfeng Lu
- Department of Biomolecular and Chemical Engineering, University of California Los Angeles, Los Angeles, California, United States of America
- California NanoSystems Institute (CNSI), University of California Los Angeles, Los Angeles, California, United States of America
| | - Masakazu Kamata
- Division of Hematology-Oncology, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America
- University of California Los Angeles AIDS Institute, Los Angeles, California, United States of America
| | - Irvin S. Y. Chen
- Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America
- University of California Los Angeles AIDS Institute, Los Angeles, California, United States of America
- * E-mail:
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Liu Y, Zhou J, Pan JA, Mabiala P, Guo D. A novel approach to block HIV-1 coreceptor CXCR4 in non-toxic manner. Mol Biotechnol 2015; 56:890-902. [PMID: 24845753 DOI: 10.1007/s12033-014-9768-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The chemokine receptor CXCR4 is one of the major coreceptors for human immunodeficiency virus type 1 (HIV-1) and considered as an important therapeutic target. Knockdown of CXCR4 by RNA interference has emerged as a promising strategy for combating HIV-1 infection. However, there is a potential drawback to this strategy as undesired side effects may occur due to the loss of natural function of CXCR4. In this study, we developed a novel approach using a single lentiviral vector to express simultaneously CXCR4 dual-shRNAs and an shRNA-resistant CXCR4 mutant possessing the most possible natural functions of CXCR4 and reduced HIV-1 coreceptor activity. Via this approach we achieved the replacement of endogenous CXCR4 by CXCR4 mutant P191A that could compensate the functional loss of endogenous CXCR4 and significant reduction of HIV-1 replication by 59.2 %. Besides, we demonstrated that construction of recombinant lentiviral vector using 2A peptide-based strategy has significant advantages over using additional promoter-based strategy, including increase of lentivirus titer and avoidance of promoter competition. Therefore, the novel approach to block HIV-1 coreceptor CXCR4 without impairing its normal function provides a new strategy for CXCR4-targeted therapeutics for HIV-1 infection and potential universal applications to knock down a cellular protein in non-toxic manner.
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Affiliation(s)
- Ye Liu
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China
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45
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Friedmann-Morvinski D, Singer O. Overexpression Models: Lentiviral Modeling of Brain Cancer. ACTA ACUST UNITED AC 2015; 3:121-39. [DOI: 10.1002/9780470942390.mo110271] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
| | - Oded Singer
- The Salk Institute for Biological Studies; La Jolla California
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46
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Yang XN, Lu YP, Liu JJ, Huang JK, Liu YP, Xiao CX, Jazag A, Ren JL, Guleng B. Piezo1 is as a novel trefoil factor family 1 binding protein that promotes gastric cancer cell mobility in vitro. Dig Dis Sci 2014; 59:1428-35. [PMID: 24798994 DOI: 10.1007/s10620-014-3044-3] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2013] [Accepted: 01/20/2014] [Indexed: 12/09/2022]
Abstract
BACKGROUND Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. AIM We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. METHODS This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. RESULTS We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. CONCLUSIONS Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.
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Affiliation(s)
- Xiao-Ning Yang
- Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, 201 Hubin South Road, Xiamen, 361004, Fujian Province, China
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47
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Tsai WH, Chang WT. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes. Methods Mol Biol 2014; 1101:321-38. [PMID: 24233788 PMCID: PMC7121774 DOI: 10.1007/978-1-62703-721-1_15] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.
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Affiliation(s)
- Wen-Hui Tsai
- Institute of Clinical Medicine, National Cheng Kung University Medical College, Taiwan, P.R. China
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48
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Ma H, Wu Y, Dang Y, Choi JG, Zhang J, Wu H. Pol III Promoters to Express Small RNAs: Delineation of Transcription Initiation. MOLECULAR THERAPY-NUCLEIC ACIDS 2014; 3:e161. [PMID: 24803291 PMCID: PMC4040628 DOI: 10.1038/mtna.2014.12] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/07/2014] [Accepted: 03/10/2014] [Indexed: 12/17/2022]
Abstract
Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to analyze small RNAs, we show that, for mouse U6 promoter, sequences immediately upstream of the putative initiation site, which is often modified to accommodate the restriction enzyme sites that enable easy cloning of small RNAs, are critical for precise transcription initiation. When the promoter is kept unmodified, transcription starts precisely from the first available A or G within the range of positions −1 to +2. In addition, we show that transcription from another commonly used pol III promoter, H1, starts at multiple sites, which results in variability at the 5′ end of the transcripts. Thus, inaccuracy of 5′ end of small RNA transcripts might be a common problem when using these promoters to express small RNAs based on currently believed concepts. Our study provides general guidelines for minimizing the variability of initiation, thereby enabling more accurate expression of small RNAs.
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Affiliation(s)
- Hongming Ma
- Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA
| | - Yonggan Wu
- Department of Genetics, Stanford University, Stanford, California, USA
| | - Ying Dang
- Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA
| | - Jang-Gi Choi
- Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA
| | - Junli Zhang
- Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA
| | - Haoquan Wu
- Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA
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Fichtner F, Urrea Castellanos R, Ülker B. Precision genetic modifications: a new era in molecular biology and crop improvement. PLANTA 2014; 239:921-39. [PMID: 24510124 DOI: 10.1007/s00425-014-2029-y] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Accepted: 01/06/2014] [Indexed: 05/26/2023]
Abstract
Recently, the use of programmable DNA-binding proteins such as ZFP/ZFNs, TALE/TALENs and CRISPR/Cas has produced unprecedented advances in gene targeting and genome editing in prokaryotes and eukaryotes. These advances allow researchers to specifically alter genes, reprogram epigenetic marks, generate site-specific deletions and potentially cure diseases. Unlike previous methods, these precision genetic modification techniques (PGMs) are specific, efficient, easy to use and economical. Here we discuss the capabilities and pitfalls of PGMs and highlight the recent, exciting applications of PGMs in molecular biology and crop genetic engineering. Further improvement of the efficiency and precision of PGM techniques will enable researchers to precisely alter gene expression and biological/chemical pathways, probe gene function, modify epigenetic marks and improve crops by increasing yield, quality and tolerance to limiting biotic and abiotic stress conditions.
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Affiliation(s)
- Franziska Fichtner
- Plant Molecular Engineering Group, Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115, Bonn, Germany
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Multiple renal cyst development but not situs abnormalities in transgenic RNAi mice against Inv::GFP rescue gene. PLoS One 2014; 9:e89652. [PMID: 24586938 PMCID: PMC3933642 DOI: 10.1371/journal.pone.0089652] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2013] [Accepted: 01/27/2014] [Indexed: 12/05/2022] Open
Abstract
In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.
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