Transgenic Organisms Research Articles

Structural study of the intrinsically disordered tardigrade damage suppressor protein (Dsup) and its complex with DNA

Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.

Cloning and Functional Analysis of a Zeaxanthin Epoxidase Gene in Ulva prolifera.

The xanthophyll cycle is a photoprotective mechanism in plants and algae, which protects the photosynthetic system from excess light damage under abiotic stress. Zeaxanthin is considered to play a pivotal role in this process. In this study, the relative content of xanthophylls was determined using HPLC-MS/MS in Ulva prolifera exposed to different salinities. The results showed that high-salt stress significantly increased the relative content of xanthophylls and led to the accumulation of zeaxanthin. It was speculated that the accumulated zeaxanthin may contribute to the response of U. prolifera to high-salt stress. Zeaxanthin epoxidase (ZEP) is a key enzyme in the xanthophyll cycle and is also involved in the synthesis of abscisic acid and carotenoids. In order to explore the biological function of ZEP, a ZEP gene was cloned and identified from U. prolifera. The CDS of UpZEP is 1122 bp and encodes 373 amino acids. Phylogenetic analysis showed that UpZEP clusters within a clade of green algae. The results of qRT-PCR showed that high-salt stress induced the expression of UpZEP. In addition, heterologous overexpression of the UpZEP gene in yeast and Chlamydomonas reinhardtii improved the salt tolerance of transgenic organisms. In conclusion, the UpZEP gene may be involved in the response of U. prolifera to high-salt stress and can improve the high-salt tolerance of transgenic organisms.

Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

월동 배추의 추위 스트레스 반응성 ERF 유전자 발굴과 활용

To mitigate plant freeze damage caused by extreme weather events, we need to elucidate the freeze resistance mechanism of freeze-tolerant plants through molecular biological approaches. Our research focuses on identifying the cold stress responsive, ERF(Ethylene-Responsive transcription Factor) genes that confer freeze tolerance in overwintering Chinese cabbage. Additionally, we aim to explore the molecular basis for conferring freezing tolerance to other organisms through genetic transformation. Expression of the ERF, cold stress responsive gene in overwintering Chinese cabbage, leads to the synthesis of AFP(Antifreeze Protein), which results in cellular freezing tolerance. We identified and sequenced functionally important ERF domain regions within the ERF genes, which we named BrERF1 and BrERF2. Their responsiveness to cold stress was confirmed through RT-PCR and qRT-PCR. Furthermore, we propose strategies to introduce BrERF1 and BrERF2 insertion plasmids into other organisms for transformation. Significant improvements in the freezing tolerance of transgenic organisms are expected to improve the productivity of these target organisms. In particular, the development of permanently transgenic plants using the BrERFs gene offers the possibility of increasing plant production under cold stress.

Climbing into their Skin to Understand Contextual Protein-Protein Associations and Localizations: Functional Investigations in Transgenic Live Model Organisms.

Borrowing some quotes from Harper Lee's novel "To Kill A Mockingbird" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.

Third International Conference “Genetically modified organisms: the history, achievements, social and environmental risks”

Third international conference “GMO: history, achievements, social and environmental risks” was held on October 3–5, 2023 in Saint Petersburg State University, Saint Petersburg, Russia in mixed format (on-site and on-line). The conference was attended by researchers from Russia, China, Germany, Spain, India, Moldova, Tadjikistan, Belarus. They discussed new genetic engineering methods, promising results and their promotion in society. A lot of interest was attracted to the sections devoted to the genome editing of various organisms and genetic engineering in agriculture. A number of reports aimed to study the fundamental problems of biology and medicine using genetically modified microorganisms and animals. The whole section was traditionally devoted to environmental studies, involving transgenic organisms, natural GMOs that arose without human intervention. Discussion of society’s attitude towards GMOs concluded the conference, where the importance of improvement of GMO diagnostics, as well as closer interaction between biologists and lawyers for improving legislation in the field of GMOs was noted.
 The conference was held with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”. The conference organizing committee expresses gratitude to the sponsors: companies Helicon and Synthol.

Noncoupled Mitochondrial Respiration as Therapeutic Approach for the Treatment of Metabolic Diseases: Focus on Transgenic Animal Models.

Mitochondrial dysfunction contributes to numerous chronic diseases, and mitochondria are targets for various toxins and xenobiotics. Therefore, the development of drugs or therapeutic strategies targeting mitochondria is an important task in modern medicine. It is well known that the primary, although not the sole, function of mitochondria is ATP generation, which is achieved by coupled respiration. However, a high membrane potential can lead to uncontrolled reactive oxygen species (ROS) production and associated dysfunction. For over 50 years, scientists have been studying various synthetic uncouplers, and for more than 30 years, uncoupling proteins that are responsible for uncoupled respiration in mitochondria. Additionally, the proteins of the mitochondrial alternative respiratory pathway exist in plant mitochondria, allowing noncoupled respiration, in which electron flow is not associated with membrane potential formation. Over the past two decades, advances in genetic engineering have facilitated the creation of various cellular and animal models that simulate the effects of uncoupled and noncoupled respiration in different tissues under various disease conditions. In this review, we summarize and discuss the findings obtained from these transgenic models. We focus on the advantages and limitations of transgenic organisms, the observed physiological and biochemical changes, and the therapeutic potential of uncoupled and noncoupled respiration.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Next generation marker-based vector concepts for rapid and unambiguous identification of single and double homozygous transgenic organisms.

For diploid model organisms, the actual transgenesis processes require subsequent periods of transgene management, which are challenging in emerging model organisms due to the lack of suitable methodology. We used the red flour beetle Tribolium castaneum, a stored-grain pest, to perform a comprehensive functional evaluation of our AClashOfStrings (ACOS) and the combined AGameOfClones/AClashOfStrings (AGOC/ACOS) vector concepts, which use four clearly distinguishable markers to provide full visual control over up to two independent transgenes. We achieved comprehensive statistical validation of our approach by systematically creating seventeen novel single and double homozygous sublines intended for fluorescence live imaging, including several sublines in which the microtubule cytoskeleton is labeled. During the mating procedures, we genotyped more than 20,000 individuals in less than 80 working hours, which corresponds to about 10 to 15 s per individual. We also confirm the functionality of our combined concept in two double transgene special cases, i.e. integration of both transgenes in close proximity on the same chromosome and integration of one transgene on the X allosome. Finally, we discuss our vector concepts regarding performance, genotyping accuracy, throughput, resource saving potential, fluorescent protein choice, modularity, adaptation to other diploid model organisms and expansion capability.

Gene Edited Seeds

The European Union is finally coming around to gene-edited seeds For a quarter century, activists such as Vandana Shiva have opposed GM crops that can help feed the world. Now, more than ever, it is time to reject their Luddite demands. In 2021, the European Union announced that it would be reviewing its 2001-era legislation governing genetically modified (GM) organisms, so as to properly account for the recent development of “gene-edited” crops, which are produced using what is known as New Genomic Techniques (NGT). To laypeople, the distinction between the various technology types may seem obscure. But from a scientific point of view, the difference is significant. Genetically modified organisms ‐ also known as transgenic organisms, or GMOs ‐ have been around since the 1990s. Many well-known GMOs have been developed by combining DNA from different types of organisms, a process that has aroused fears of “frankenfoods” (a pejorative term coined three decades ago by Boston College professor Paul Lewis, who wrote that if they want to sell us Frankenfood, perhaps it is time to gather the villagers, light some torches and head to the castle. By contrast, NGTs allow scientists to manipulate specific regions of a genome directly ‐ so as to reduce the need for pesticides, resist disease, boost yields, or enhance resilience in the face of climate change ‐ without importing genetic material from outside sources.

Plant Promoters: Their Identification, Characterization, and Role in Gene Regulation.

One of the strategies to overcome diseases or abiotic stress in crops is the use of improved varieties. Genetic improvement could be accomplished through different methods, including conventional breeding, induced mutation, genetic transformation, or gene editing. The gene function and regulated expression through promoters are necessary for transgenic crops to improve specific traits. The variety of promoter sequences has increased in the generation of genetically modified crops because they could lead to the expression of the gene responsible for the improved trait in a specific manner. Therefore, the characterization of the promoter activity is necessary for the generation of biotechnological crops. That is why several analyses have focused on identifying and isolating promoters using techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR), genetic libraries, cloning, and sequencing. Promoter analysis involves the plant genetic transformation method, a potent tool for determining the promoter activity and function of genes in plants, contributing to understanding gene regulation and plant development. Furthermore, the study of promoters that play a fundamental role in gene regulation is highly relevant. The study of regulation and development in transgenic organisms has made it possible to understand the benefits of directing gene expression in a temporal, spatial, and even controlled manner, confirming the great diversity of promoters discovered and developed. Therefore, promoters are a crucial tool in biotechnological processes to ensure the correct expression of a gene. This review highlights various types of promoters and their functionality in the generation of genetically modified crops.

Invasive Potential and Regulation Experience of Using Transgenic Organisms in Aquaculture by Way of Example of Atlantic Salmon

Invasive Potential and Regulation Experience of Using Transgenic Organisms in Aquaculture by Way of Example of Atlantic Salmon

Pharmaceutical and Biopharmaceuticals Industries: Revolutionizing Healthcare

The pharmaceutical and biopharmaceuticals industries play a crucial role in revolutionizing healthcare by developing and delivering innovative medicines and therapies. These industries combine cutting-edge scientific research, advanced technologies, and rigorous regulatory processes to address a wide range of diseases and improve patients' lives. In this article, we will explore the key aspects of the pharmaceutical and biopharmaceuticals sectors and their impact on healthcare. Pharmaceuticals, a substance used in the diagnosis, treatment, or prevention of sickness and for restoring, correcting, or editing natural functions. A biopharmaceuticals (biological or biologic) comprising sugars, proteins, nucleic acids, living cells, or tissues is a medicinal product manufactured in extracted or semi-synthesized from natural sources like humans, animals, or microorganisms. Different from ordinary pills synthesized from chemical processes, we derive most biopharmaceuticals merchandise from natural processes alongside the extraction from residing buildings or the manufacturing with the resource of recombinant DNA used sciences. Transgenic organisms, especially plants, animals, or microorganisms that have been genetically modified, are doubtlessly used to produce biopharmaceuticals

In Vitro Fertilization of the African Turquoise Killifish Nothobranchius furzeri.

The ability to perform in vitro fertilization, together with sperm cryopreservation, greatly facilitates the long-term laboratory maintenance of wild-type and transgenic model organisms and helps prevent genetic drift. It is also useful in cases where reproduction may be compromised. In this protocol, we present a method for in vitro fertilization of the African Turquoise killifish Nothobranchius furzeri that is compatible with the use of fresh or cryopreserved sperm.

Lithbea, a New Domain Outside the Tree of Life

At this time when the development of synthetic biology and artificial intelligence are changing the world around us, philosophers and scientists, first of all, must converge to analyze the present and predict the ethical-social consequences and biological dangers associated with new “living entities” that are not the result of the natural evolutionary process. As synthetic/artificial life forms (xenobots, robots, transgenic organisms, etc.) become more and more abundant and sophisticated, it seems first of all necessary to bring some order to all this new biodiversity, establishing what is alive and what is not, and analyzing the consequences of this incessant creative activity. Here I intend to organize all these human-made entities and clarify their status as living beings or artificial elements, leaving the door open to an uncertain future in which we will be able to see how “the artificial” and “the natural” could merge to originate something different from everything known. Accordingly, I propose the creation of a new domain, Lithbea, which includes all synthetic and artificial entities within a new kingdom called Humade (derived from human-made). I have also included viruses in a new realm, the Viral kingdom, because they were excluded from the classical three-domain tree of life despite playing a fundamental role in the evolution of biodiversity on Earth. Finally, I make a brief comment on the unpredictability of the unknown, the implications of this new landscape of biodiversity, and the uncertain future of all these advances.

СОДЕРЖАНИЕ НЕЙРОГЛОБИНА В СТРУКТУРАХ ГОЛОВНОГО МОЗГА КРЫСЫ ПРИ СУБТОТАЛЬНОЙ ИШЕМИИ

Neuroglobin (Ngb) is a member of the globin family. Like other globin proteins, it is involved in the maintenance of oxygen homeostasis. Ngb distribution in the normal brain is well known, but its response to pathological injury, such as cerebral ischemia, has not yet been adequately elucidated. One primary reason for this is that ischemic lesions in cerebral tissues have mostly been studied in transgenic organisms. In addition, the available data on Ngb content are limited to a small number of brain structures. This article examines the patterns of changes in Ngb immunoreactivity in neurons from different parts of the rat brain after subtotal cerebral ischemia of varying duration. An immunohistochemical study of Ngb content in 25 brain structures of white male Wistar rats exposed to 30-min and 3-h subtotal cerebral ischemia was performed. A decrease in Ngb content in all structures (especially 3 h after the ischemia onset and in the phylogenetically older parts), temporal mediation, and dependence on the phylogenetic age were revealed. The obtained results further decipher the correlation between the changes in Ngb content and the degree of cerebral ischemic damage, which is necessary to clarify the functions of the studied protein.

Toxicity, Transgenics, and the Flesh of Fiction in Samanta Schweblin

Toxins lurk in the soy fields around a small Argentine town in Samanta Schweblin’s 2014 novel Distancia de rescate. On the surface, Schweblin’s novel appears to use supernatural phenomena to criticize industrial soy production and its use of toxic herbicides. Current criticism, in turn, sees the novel as an extension of the Latin American fantastic, filtered through an ecocritical or Anthropocene lens. However, I argue that Schweblin’s novel, read alongside her short story “Conservas,” portrays a more complex relationship between flesh and the environment. Drawing on the ideas of Maurice Merleau-Ponty, I argue that the chiasm, or crossing over, of flesh mimics the structure of transgenic organisms so that the characters resemble genetically modified soy. I show how Schweblin portrays flesh as reversible, interconnected, and increasingly intoxicated. Following Julia Kristeva and Jacques Derrida, she uses an abject counteraesthetic to blend nature and artifice and to question traditional distinctions between human and environment, subject and object that emerge from Kantian philosophy and continue into the aesthetic tradition of the sublime. In their place, Schweblin suggests an already intoxicated flesh that forms the basis of the categories of subject and object and an abject, toxic sublime, present in flesh and fiction.

The Effect of Mating Complexity on Gene Drive Dynamics.

AbstractGene drive technology promises to deliver on some of the global challenges humanity faces today in health care, agriculture, and conservation. However, there is a limited understanding of the consequences of releasing self-perpetuating transgenic organisms into wild populations under complex ecological conditions. In this study, we analyze the impact of three such complexities-mate choice, mating systems, and spatial mating network-on the population dynamics for two distinct classes of modification gene drive systems. All three factors had a high impact on the modeling outcome. First, we demonstrate that distortion-based gene drives appear to be more robust against mate choice than viability-based gene drives. Second, we find that gene drive spread is much faster for higher degrees of polygamy. Including a fitness cost, the drive is fastest for intermediate levels of polygamy. Finally, the spread of a gene drive is faster and more effective when the individuals have fewer connections in a spatial mating network. Our results highlight the need to include mating complexities when modeling the properties of gene drives, such as release thresholds, timescales, and population-level consequences. This inclusion will enable a more confident prediction of the dynamics of engineered gene drives and possibly even inform about the origin and evolution of natural gene drives.

MINERVA: A CubeSat for demonstrating DNA damage mitigation against space radiation in C. elegans by using genetic modification

The ideas of deep-space human exploration, interplanetary travel, and space civilizations are becoming a reality. However, numerous hindrances remain standing in the way of accomplishing these feats, one of which is space ionizing radiation. Space ionizing radiation has become the most hazardous health risk for long-term human space exploration, as it can induce chromosomal damage and epigenetic changes. The Minerva mission aims to demonstrate cutting-edge technology to inhibit DNA damage against deep-space radiation exposure by using genetic modification. The concept of the experiment is to transform a creature with radiation intolerance into a transgenic organism that is radiation-tolerant. In this mission, Caenorhabditis elegans (C. elegans) will be genetically engineered with a protein-coding gene associated with DNA damage protection called damage suppressor (Dsup). Dsup is a nucleosome-binding protein from the tardigrade Ramazzottius varieornatus that has a unique ability to prevent DNA damage. This paper describes the feasibility of Minerva CubeSat, which will venture out to cis-lunar orbit with a biosensor payload capable of sustaining and culturing C. elegans under space environment conditions for 4 months. The mission will set in motion a paradigm shift corresponding to future space medicines and how they will be developed in the future, introducing a platform suitable for future experiments in the fields of space biology. Ultimately, the paramount objective of Minerva will be to test the limits of genetic engineering and incorporate it into the arduous journey of human perseverance to overcome the boundaries of space exploration—a vital step in making Mars colonization safe.

A Rapid and Noninvasive Method That Extracts Polymerase Chain Reaction-Ready Genomic DNA from Adult Zebrafish.

Genotyping usually entails analysis of the products of polymerase chain reaction (PCR) carried out with genomic DNA (gDNA) as template, and is employed for validation of mutant or transgenic organisms. For genotyping of adult zebrafish, gDNA is often extracted from clipped caudal fin or skin mucus through either alkaline lysis using NaOH or proteinase K (PK) treatment. Further purification of the gDNA using ethanol precipitation was optional. To develop a rapid and noninvasive method that extracts PCR-ready gDNA from adult zebrafish, we combined skin swabbing with PK treatment and demonstrated its efficiency. This method could be applied to a wide range of fish.