Genetic Manipulation Technologies Research Articles

Gene editing technology and its recent progress in disease therapy

Gene editing is a genetic manipulation technology which utilizes bacterial nucleases to accurately and efficiently modify DNA or RNA. Gene editing has broad applications in basic research, breeding, and drug screening, and it is gaining validity and applicability to the therapy of many diseases especially genetic-based disease. In this review, we summarize the development of gene editing technology, its different strategies and applications in the treatment of disease, and the research of gene editing therapy for genetic diseases (including base editor and epigenetic regulation) in the treatment of disorders and diseases of the blood system, liver, muscle and nervous system. Finally, we discuss the future development prospects of gene editing therapy.

International policy and ethics in human genome editing: An overview of position statements

Background & Aim For more than two decades, scientists have been developing genetic engineering and manipulation technologies for industrial, agricultural and medical purposes. However, until recently, clinician-researchers have had with limited success in translating them into useful clinical applications. The expansion of gene modifying technologies in translational research is accelerating with the availability of easily accessible and inexpensive modification tools, such as the CRISPR-Cas9 system. This simple, cost-efficient, cut-and-paste tool has given rise to the moniker ‘gene editing’. Whether gene editing raises any fundamentally new or different ethical issues than those raised with earlier gene modifying technologies is unclear; especially around heritable germline modifications in humans. Yet, the controversy that exploded in 2018 around revelations that a Chinese scientist had (apparently) modified the CCR5 gene of twin baby girls suggests that, at least, some of those issues remain unresolved, and that any social consensus to move forward with this technology in germline applications remains far away. Methods, Results & Conclusion This paper provides an overview of the international science policy landscape emergent since CRISPR-Cas9 was first used to edit the genes of human embryos in 2015. I summarise key ethical and policy positions issued by major regulatory institutions in the US and the EU, starting with the 2015 Summary Statement of the International Summit on Human Genome Editing, which was subsequently reiterated by the organisers of the second meeting held in November 2018 where details of the children born with modified genes were presented. Positions generally held that the use of genome modifying technologies in developing somatic therapies was ethically acceptable and ought to be encouraged. However, while they agree that it remains technically premature to develop this technology for germline therapies, there are some discrepancies over the ethics of eventually doing so once safety barriers are overcome. I discuss these positions with reference to ethical frameworks that aim to encourage responsibility in the clinical translation of genome modifying technologies, and conclude with mention of guidance being developed at the World Health Organisation and the WHO Collaborating Centre for Bioethics in Singapore.

AMPA Receptor Trafficking for Postsynaptic Potentiation.

Long-term potentiation (LTP) of excitatory synaptic strength, which has long been considered a synaptic correlate for learning and memory, requires a fast recruitment of additional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs) to the postsynaptic sites. As cell biological concepts have been applied to the field and genetic manipulation and microscopic imaging technologies have been advanced, visualization of the trafficking of AMPARs to synapses for LTP has been investigated intensively over the last decade. Recycling endosomes have been reported as intracellular storage organelles to supply AMPARs for LTP through the endocytic recycling pathway. In addition, exocytic domains in the spine plasma membrane, where AMPARs are inserted from the intracellular compartment, and nanodomains, where diffusing AMPARs are trapped and immobilized inside synapses for LTP, have been described. Furthermore, cell surface lateral diffusion of AMPARs from extrasynaptic to synaptic sites has been reported as a key step for AMPAR location to the synaptic sites for LTP. This review article will discuss recent findings and views on the reservoir(s) of AMPARs and their trafficking for LTP expression by focusing on the exocytosis and lateral diffusion of AMPARs, and provide some future directions that need to be addressed in the field of LTP.

Cre Driver Mice Targeting Macrophages.

The Cre/loxP system is a widely applied technology for site-specific genetic manipulation in mice. This system allows for deletion of the genes of interest in specific cells, tissues, and whole organism to generate a diversity of conditional knockout mouse strains. Additionally, the Cre/loxP system is useful for development of cell- and tissue-specific reporter mice for lineage tracing, and cell-specific conditional depletion models in mice. Recently, the Cre/loxP technique was extensively adopted to characterize the monocyte/macrophage biology in mouse models. Compared to other relatively homogenous immune cell types such as neutrophils, mast cells, and basophils, monocytes/macrophages represent a highly heterogeneous population which lack specific markers or transcriptional factors. Though great efforts have been made toward establishing macrophage-specific Cre driver mice in the past decade, all of the current available strains are not perfect with regard to their depletion efficiency and targeting specificity for endogenous macrophages. Here we overview the commonly used Cre driver mouse strains targeting macrophages and discuss their major applications and limitations.

Cassava Mosaic and Brown Streak Diseases: Current Perspectives and Beyond.

Cassava is the fourth largest source of calories in the world but is subject to economically important yield losses due to viral diseases, including cassava brown streak disease and cassava mosaic disease. Cassava mosaic disease occurs in sub-Saharan Africa and the Asian subcontinent and is associated with nine begomovirus species, whereas cassava brown streak disease has to date been reported only in sub-Saharan Africa and is caused by two distinct ipomovirus species. We present an overview of key milestones and their significance in the understanding and characterization of these two major diseases as well as their associated viruses and whitefly vector. New biotechnologies offer a wide range of opportunities to reduce virus-associated yield losses in cassava for farmers and can additionally enable the exploitation of this valuable crop for industrial purposes. This review explores established and new technologies for genetic manipulation to achieve desired traits such as virus resistance.

Rapid inducible protein displacement in Plasmodiumin vivo and in vitro using knocksideways technology.

A deeper understanding of the biology of the Plasmodium parasite is essential in order to identify targets for interventions, with the ultimate aim of eliminating malaria. Determining the function(s) of essential proteins in Plasmodium has, until recently, been hampered by the lack of efficient conditional systems to abrogate proteins. We report the adaptation of a conditional technology, knocksideways (KS), for use in Plasmodium berghei, which can potentially rapidly inactivate proteins of interest through relocalisation. The system is induced using rapamycin, which allows for KS both in vitro and in vivo and is effective more rapidly than any other reported system. KS utilises pairs of fluorescent tags that facilitate live imaging and allows for rapid confirmation of efficient protein redistribution on live parasites, allowing for streamlined workflows. We demonstrate the characteristics of the system using transgenically expressed cytoplasmic GFP and provide proof of principle by inducibly redistributing a number of proteins with different native, subcellular locations. We also demonstrate that KS can be applied to both mammalian and insect stages of Plasmodium. KS expands the range of (conditional) technologies for genetic manipulation of malaria parasites and offers the potential to be further developed for medium throughput phenotype screens.

Regenerasi Jeruk Siam melalui Embriogenesis Somatik

<p>The Regenerated of Siam Tangerin through Somatic<br />Embryogenesis. Ali Husni, Agus Purwito, Ika Mariska,<br />and Sudarsono. Somatic embryogenesis occurs in most<br />plants that are cultured on a suitable medium in vitro.<br />Somatic embryo may arise from single cells and the embryogenic<br />cells are widely applicable in plant propagation,<br />genetic manipulation and transgenic technologies. The<br />present study was carried out to develop an effective<br />somatic embryogenesis technique to regenerate Siam<br />tangerine plants. Materials used in this study were nucellar<br />tissues of young fruits (30-90 days post anthesis). Induction<br />of embryogenic calli was done by culturing the tissues on<br />three different basal media (MS, MW and MT). Embryo<br />maturation was done on the MW medium + ABA (0; 0.1; 0.3;<br />dan 0.5 mg/l), while germination to plantlet development<br />was done on WS medium + GA3 (0, 0.1, 0.3, and 0.5 mg/l).<br />The results showed that among the three media, MW was<br />the best medium for callus induction from nucellar tissues<br />with a success rates 98% for Simadu and 100% for<br />Pontianak. The best maturation of embryo somatic was<br />done on MW medium + ABA 0.5 mg/l with success rates<br />99%, while the best medium for germination and development<br />into planlets was MW medium + 0.5 mg/l GA3 with a<br />success rate 58%.</p>

Advances and perspectives in the application of CRISPR/Cas9 in insects.

Insects compose more than half of all living organisms on earth, playing essential roles in global ecosystems and forming complex relationships with humans. Insect research has significant biological and practical importance. However, the application of genetic manipulation technology has long been restricted to several model insects only, such as gene knockout in Drosophila, which has severely restrained the development of insect biology research. Recently, with the increase in the release of insect genome data and the introduction of the CRISPR/Cas9 system for efficient genetic modification, it has been possible to conduct meaningful functional studies in a broad array of insect species. Here, we summarize the advances in CRISPR/Cas9 in different insect species, discuss methods for its promotion, and consider its application in future insect studies. This review provides detailed information about the application of the CRISPR/Cas9 system in insect research and presents possible ways to improve its use in functional studies and insect pest control.

Genetic manipulation of putrescine biosynthesis reprograms the cellular transcriptome and the metabolome.

BackgroundWith the increasing interest in metabolic engineering of plants using genetic manipulation and gene editing technologies to enhance growth, nutritional value and environmental adaptation, a major concern is the potential of undesirable broad and distant effects of manipulating the target gene or metabolic step in the resulting plant. A comprehensive transcriptomic and metabolomic analysis of the product may shed some useful light in this regard. The present study used these two techniques with plant cell cultures to analyze the effects of genetic manipulation of a single step in the biosynthesis of polyamines because of their well-known roles in plant growth, development and stress responses.ResultsThe transcriptomes and metabolomes of a control and a high putrescine (HP) producing cell line of poplar (Populus nigra x maximowiczii) were compared using microarrays and GC/MS. The HP cells expressed an ornithine decarboxylase transgene and accumulated several-fold higher concentrations of putrescine, with only small changes in spermidine and spermine. The results show that up-regulation of a single step in the polyamine biosynthetic pathway (i.e. ornithine → putrescine) altered the expression of a broad spectrum of genes; many of which were involved in transcription, translation, membrane transport, osmoregulation, shock/stress/wounding, and cell wall metabolism. More than half of the 200 detected metabolites were significantly altered (p ≤ 0.05) in the HP cells irrespective of sampling date. The most noteworthy differences were in organic acids, carbohydrates and nitrogen-containing metabolites.ConclusionsThe results provide valuable information about the role of polyamines in regulating nitrogen and carbon use pathways in cell cultures of high putrescine producing transgenic cells of poplar vs. their low putrescine counterparts. The results underscore the complexity of cellular responses to genetic perturbation of a single metabolic step related to nitrogen metabolism in plants. Combined with recent studies from our lab, where we showed that higher putrescine production caused an increased flux of glutamate into ornithine concurrent with enhancement in glutamate production via additional nitrogen and carbon assimilation, the results from this study provide guidance in designing transgenic plants with increased nitrogen use efficiency, especially in plants intended for non-food/feed applications (e.g. increased biomass production for biofuels).Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0796-2) contains supplementary material, which is available to authorized users.

Reload approach of human dignity in the realm of bioethics

Background and Aim: Bioethics as a new branch of normative ethics and applied to the basic strategies in the face of questions and answers explaining the challenges posed by the emergence of new technologies, biomedical deals. Implementation issues such as human cloning, stem cells, shimmer and demonization organisms, genetic manipulation and gene-related technologies and biomedical experiments on human subjects, is subject to bioethics studies. In the meantime, Human Dignity as the source of all human rights has an important place in the field of bioethics and biomedical technology to permit or prohibit any behavior or action in this area. Materials and Methods: In this article with the use of previous studies about Human Dignity and the search keywords in the origin sources, tried to analyze and assesse the place of human dignity in the context of the implementation of Bioethics. Ethical Consideration: Principles of research ethics were observed in searches and cite texts and primary sources. Finding: In this brief article the different approaches of human dignity in connection with bioethics have been studied and evaluated: assessment of religiosity readings that reflects the characteristics and traits of inherent dignity of the human being and attributes of Allah the way He sees; exposition of philosophical interpretations ranging from the tradition of Kantian deontological, utilitarian approach and the approach of a naturalist's among the subjects, that in this study descriptive, analytical, and criticized the accuracy and purpose of issue of why the prohibition and criminalization in the areas of bioethical issues. Conclution: In the final classification and the integration of various approaches, the importance of human dignity as the source of all human rights has been asserted and any restriction on the scope of bioethics must be in the light of observation and respection to the dignity of the human identity. Please cite this article as: Rahbarpour MR. Reload approach of human dignity in the realm of bioethics. Iran J Bioethics 2017; 6(22): 19-31.

A strong root-specific expression system for stable transgene expression in bread wheat.

A strong, stable and root-specific expression system was developed from a rice root-specific GLYCINE - RICH PROTEIN 7 promoter for use as an enabling technology for genetic manipulation of wheat root traits. Root systems play an important role in wheat productivity. Genetic manipulation of wheat root traits often requires a root-specific or root-predominant expression system as an essential enabling technology. In this study, we investigated promoters from rice root-specific or root-predominant expressed genes for development of a root expression system in bread wheat. Transient expression analysis using a GREEN FLUORESCENT PROTEIN (GFP) reporter gene driven by rice promoters identified six promoters that were strongly expressed in wheat roots. Extensive organ specificity analysis of three rice promoters in transgenic wheat revealed that the promoter of rice GLYCINE-RICH PROTEIN 7 (OsGRP7) gene conferred a root-specific expression pattern in wheat. Strong GFP fluorescence in the seminal and branch roots of wheat expressing GFP reporter driven by the OsGRP7 promoter was detected in epidermal, cortical and endodermal cells in mature parts of the root. The GFP reporter driven by the promoter of rice METALLOTHIONEIN-LIKE PROTEIN 1 (OsMTL1) gene was mainly expressed in the roots with essentially no expression in the leaf, stem or seed. However, it was also expressed in floral organs including glume, lemma, palea and awn. In contrast, strong expression of rice RCg2 promoter-driven GFP was found in many tissues. The GFP expression driven by these three rice promoters was stable in transgenic wheat plants through three generations (T1-T3) examined. These data suggest that the OsGRP7 promoter can provide a strong, stable and root-specific expression system for use as an enabling technology for genetic manipulation of wheat root traits.

Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies.

Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high-quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 kb, 3.4 Mb) and Papilio machaon (contig and scaffold N50: 81 kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations, and adult and larval colour patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony and frizzled. Our results provide valuable genomic and technological resources for butterflies and unlock their potential as a genetic model system.

Application of genetically engineered mice model in the research of gastrointestinal tumor

The gene knockout is based on the DNA homologous recombination and embryonic stem cell technology. It is an experimental method for modification of specific gene loci, which is one of the most direct means to clarifying gene functions. The similarity of genomes between mouse and human is as high as up to 95%. The life cycle of mice is relatively short, and easy to breed with stronger reproductive capacity. Therefore, the knockout mice are suitable for observing the whole process of disease pathogenesis. With genetic manipulation technology, researchers can produce the simulations of phenotypic effect by human genetic variation. This article focuses on genetically engineered mice models and their application in gastrointestinal cancer.

Gene targeting technologies in rats: Zinc finger nucleases, transcription activator‐like effector nucleases, and clustered regularly interspaced short palindromic repeats

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed.

One mouse, one patient paradigm: New avatars of personalized cancer therapy

Over the last few decades, study of cancer in mouse models has gained popularity. Sophisticated genetic manipulation technologies and commercialization of these murine systems have made it possible to generate mice to study human disease. Given the large socio-economic burden of cancer, both on academic research and the health care industry, there is a need for in vivo animal cancer models that can provide a rationale that is translatable to the clinic. Such a bench-to-bedside transition will facilitate a long term robust strategy that is economically feasible and clinically effective to manage cancer. The major hurdles in considering mouse models as a translational platform are the lack of tumor heterogeneity and genetic diversity, which are a hallmark of human cancers. The present review, while critical of these pitfalls, discusses two newly emerging concepts of personalized mouse models called “Mouse Avatars” and Co-clinical Trials. Development of “Mouse Avatars” entails implantation of patient tumor samples in mice for subsequent use in drug efficacy studies. These avatars allow for each patient to have their own tumor growing in an in vivo system, thereby allowing the identification of a personalized therapeutic regimen, eliminating the cost and toxicity associated with non-targeted chemotherapeutic measures. In Co-clinical Trials, genetically engineered mouse models (GEMMs) are used to guide therapy in an ongoing human patient trial. Murine and patient trials are conducted concurrently, and information obtained from the murine system is applied towards future clinical management of the patient’s tumor. The concurrent trials allow for a real-time integration of the murine and human tumor data. In combination with several molecular profiling techniques, the “Mouse Avatar” and Co-clinical Trial concepts have the potential to revolutionize the drug development and health care process. The present review outlines the current status, challenges and the future potential of these two new in vivo approaches in the field of personalized oncology.

Structural and biomechanical responses of osseous healing: a novel murine nonunion model

BackgroundUnderstanding the biological mechanisms of why certain fractures are at risk for delayed healing or nonunion requires translational animal models that take advantage of transgenic and other genetic manipulation technologies. Reliable murine nonunion models can be an important tool to understand the biology of nonunion. In this study, we report the results of a recently established model for creating critical defects that lead to atrophic nonunions based on a unique fracture fixation technique.Materials and methodsSubcritical (0.6 mm long) and critical (1.6 mm long) defects were created in femurs of 10-week-old double transgenic (Col1/Col2) mice and stabilized using a custom-designed plate and four screws. Four groups were used: normal, sham, subcritical, and critical. Histology (n = 3 for each group) was analyzed at 2 and 5 weeks, and micro-computed tomography (μCT) and torsional biomechanics (n = 12 for each group) were analyzed at 5 weeks.ResultsSubcritical defects showed healing at 2 weeks and were completely healed by 5 weeks, with biomechanical properties not significantly different from normal controls. However, critical defects showed no healing by histology or μCT. These nonunion fractures also displayed no torsional stiffness or strength in 10 of 12 cases.ConclusionsOur murine fracture model creates reproducible and reliable nonunions and can serve as an ideal platform for studying molecular pathways to contrast healing versus nonhealing events and for evaluating innovative therapeutic approaches to promote healing of a challenging osseous injury.Electronic supplementary materialThe online version of this article (doi:10.1007/s10195-013-0269-4) contains supplementary material, which is available to authorized users.

The Fate of an Engineered Planet

The article presents fictitious accounts of possible technological innovations that might be designed to address rising global temperatures in the 100 years following 2013. According to the accounts, solar geoengineering could be used to spray particles into the atmosphere and deflect solar radiation. The possibility of an international climate treaty to reduce greenhouse gas emissions is mentioned. Other topics include international relations, a fictitious U.S. drought, and genetic manipulation technologies.

Failure of homologous synapsis and sex-specific reproduction problems.

The prophase of meiosis I ensures the correct segregation of chromosomes to each daughter cell. This includes the pairing, synapsis, and recombination of homologous chromosomes. A subset of chromosomal abnormalities, including translocation and inversion, disturbs these processes, resulting in the failure to complete synapsis. This activates the meiotic pachytene checkpoint, and the gametes are fated to undergo cell cycle arrest and subsequent apoptosis. Spermatogenic cells appear to be more vulnerable to the pachytene checkpoint, and male carriers of chromosomal abnormalities are more susceptible to infertility. In contrast, oocytes tend to bypass the checkpoint and instead generate other problems, such as chromosome imbalance that often leads to recurrent pregnancy loss in female carriers. Recent advances in genetic manipulation technologies have increased our knowledge about the pachytene checkpoint and surveillance systems that detect chromosomal synapsis. This review focuses on the consequences of synapsis failure in humans and provides an overview of the mechanisms involved. We also discuss the sexual dimorphism of the involved pathways that leads to the differences in reproductive outcomes between males and females.

Cell culture models and animal models for studying the patho-physiological role of renal aquaporins.

Aquaporins (AQPs) are key players regulating urinary-concentrating ability. To date, eight aquaporins have been characterized and localized along the nephron, namely, AQP1 located in the proximal tubule, thin descending limb of Henle, and vasa recta; AQP2, AQP3 and AQP4 in collecting duct principal cells; AQP5 in intercalated cell type B; AQP6 in intercalated cells type A in the papilla; AQP7, AQP8 and AQP11 in the proximal tubule. AQP2, whose expression and cellular distribution is dependent on vasopressin stimulation, is involved in hereditary and acquired diseases affecting urine-concentrating mechanisms. Due to the lack of selective aquaporin inhibitors, the patho-physiological role of renal aquaporins has not yet been completely clarified, and despite extensive studies, several questions remain unanswered. Until the recent and large-scale development of genetic manipulation technology, which has led to the generation of transgenic mice models, our knowledge on renal aquaporin regulation was mainly based on in vitro studies with suitable renal cell models. Transgenic and knockout technology approaches are providing pivotal information on the role of aquaporins in health and disease. The main goal of this review is to update and summarize what we can learn from cell and animal models that will shed more light on our understanding of aquaporin-dependent renal water regulation.

Progress in The Genetic Manipulation Technology of Embryonic Stem Cells in Rats

The successful establishment of rat ES cell lines enables possible genetic manipulation in rats.Using homologous recombination to modify ES cell genes provides a foundation to temporal and tissue-specific knockout rats.The author introduces the establishment process of rat ES cell,summarizes its culture and identification skills,and analyzes the weakness and strength of various rat knockout techniques.In the context of increasing research on stem cell,knockout technology based on rat ES cells,which allows the most efficient modification of specific genes,shall contribute more to elucidate gene function,dissect the genetic mechanisms of human diseases and identify potential targets for drug development.