Use Of Transgenic Animals Research Articles

TT2023 meeting report on the 18th Transgenic Technology meeting in Houston, United States.

The 18th Transgenic Technology Meeting, held in Houston, Texas from November 12-15, 2023, was a vibrant international forum. It brought together nearly 400 delegates to discuss advances in transgenic technologies and the science these technologies support. Among them were 329 in-person and 70 remote delegates, representing 26 countries from 5 continents. The event, hosted by the International Society for Transgenic Technologies (ISTT), was set against the backdrop of the Hyatt Regency's panoramic views, reflecting the innovative spirit of the conference. A notable precursor to the main conference was the Allele Design Pre-conference Workshop, which fostered in-depth discussions on state-of-the-art methodologies. The main conference encompassed ten sessions, delving into diverse topics from Precision Animal Models of Human Disease to the use of transgenic animals in Space Biology. Eighty posters provided for a lively exchange of ideas, while the ISTT Prize and other awards highlighted the event's commitment to excellence. Beyond the conference halls, attendees had the opportunity to venture into Houston's Museum District, home to 19 museums in the downtown area, or indulge in unique dining experiences.

The role of nerve growth factor in traumatic brain injury

Nerve growth factor (NGF), a crucial member of neurotrophic factors (NTFs), plays a significant role in various diseases, including spinal cord injury, brain ischemia, and Alzheimer's disease. Research indicates NGF's pivotal involvement in traumatic brain injury (TBI). Animal models such as fluid percussion and weight-drop impact models were employed to explore TBI pathology, pathophysiology, and treatment. In these models, NGF, in correlation with other NTFs, significantly increased post-TBI, sustained by glucocorticoids and neuroactive steroid progesterone but inhibited by hypothermia. NGF exhibits neuroprotective effects in TBI, attenuating brain edema, activating antioxidants, inhibiting neuron loss, and alleviating amyloid β42 (Aβ42) deposition. Early up-regulation of NGF in children's cerebral spinal fluid (CSF) 2 hours post-TBI correlates with injury severity and improved functional outcomes. Various TBI treatments, such as marrow stem cells (MSCs), NT2N implantation, and hyperbaric oxygen (HBO) treatment, correlate with changes in NGF expression. Additionally, NGF and Brain-Derived Neurotrophic Factor (BDNF) promote MSC migration and differentiation in vivo. Despite two decades of research on NGF in TBI models, there is a lack of persuasive evidence regarding its function and mechanism. The limited use of transgenic animals or RNA interference for NGF hinders investigations. Effective treatment of TBI with NGF-related strategies is still a significant challenge.

The mTOR pathway and transgenic animals with deletion of the TSC gene in the regeneration of the nervous system and selected models of sciatic nerve damage

Traumatic damage to the nervous system has been a common occurrence for years, reducing patients' quality of life. The mammalian target of rapamycin (mTOR) pathway plays a key role in nervous system physiology, including by controlling nerve cell survival and differentiation. Excessive activation of the mTOR pathway leads to an increase in cell cycle protein activity and apoptosis of nerve cells. Moreover, current findings suggest the involvement of the mTOR pathway in neuroplasticity. The use of transgenic animals with deletion of the TSC gene as well as various models of sciatic nerve damage, allows activation of the mTOR pathway. Currently, the results confirm that inactivation of point mutations in TSC-1 or TSC-2 genes, activates the canonical signaling pathway of the mTORC-1 complex, in turn, reactivation of the mTORC-1 pathway through the absence of the TSC-1 gene in mature neurons induces axonal regeneration. Dysfunction of the mTORC-1 pathway in Schwann cells (SC) inhibits myelination of nerve fibers. The aim of the present study is to understand the physiology and role of the mTOR pathway as well as to demonstrate the impact of TSC gene deletion in the regeneration of the nervous system. Current research on the activity of the mTOR pathway may provide new strategies to intensify peripheral nerve regeneration.

A Simple In-Vivo Method for Evaluation of Antibiofilm and Wound Healing Activity Using Excision Wound Model in Diabetic Swiss Albino Mice

The study developed a simple and inexpensive method to induce biofilm formation in-vivo for the evaluation of the antibiofilm activity of pharmacological agents using Swiss albino mice. Animals were made diabetic using streptozocin and nicotinamide. A cover slip containing preformed biofilm along with MRSA culture was introduced into the excision wound in these animals. The method was effective in developing biofilm on the coverslip after 24 h incubation in MRSA broth which was confirmed by microscopic examination and a crystal violet assay. Application of preformed biofilm along with microbial culture induced a profound infection with biofilm formation on excision wounds in 72 h. This was confirmed by macroscopic, histological, and bacterial load determination. Mupirocin, a known antibacterial agent effective against MRSA was used to demonstrate antibiofilm activity. Mupirocin was able to completely heal the excised wounds in 19 to 21 days while in the base-treated group, healing took place between 30 and 35 days. The method described is robust and can be reproduced easily without the use of transgenic animals and sophisticated methods such as confocal microscopy.

How to Generate a Vascular-Labelled Transgenic Zebrafish Model to Study Tumor Angiogenesis and Extravasation.

The use of transgenic animals carrying exogenous DNA integrated in their genome is a routine in modern-day laboratories. Nowadays, the zebrafish system represents the most useful tool for transgenesis studies mainly due to easy accessibility and manipulation of the eggs, which are produced in high numbersand overa relatively short generation time. The zebrafish transgenic technology is very straightforward when coupled with angiogenesis studies allowing easy in vivo observation of the vertebrate embryonic vasculature. Here, we describe the most common technique to generate vascular-labelled transgenic zebrafish embryos and their applications to study tumor angiogenesis and visualize tumor extravasation.

Goodnight, astrocyte: waking up to astroglial mechanisms in sleep.

Astrocytes mediate many important aspects of neural homeostasis, but until recently, their role in sleep was largely unknown. The situation has dramatically changed in the last decade. The use of transgenic animals, optogenetics, chemogenetics, brain imaging and sophisticated molecular assays has led to exciting discoveries. Astrocytes dynamically change their activity across the sleep-wake cycle and may encode sleep need via changes in intracellular signalling pathways. Astrocytes also exocytose/secrete sleep-inducing molecules which modulate brain activity, sleep architecture and sleep regulation. Many of these observations have been made in mice and Drosophila melanogaster, indicating that astroglial sleep mechanisms are evolutionarily conserved. We review recent findings and discuss future directions.

Mechanistic Insight into Orthodontic Tooth Movement Based on Animal Studies: A Critical Review.

Alveolar bone remodeling in orthodontic tooth movement (OTM) is a highly regulated process that coordinates bone resorption by osteoclasts and new bone formation by osteoblasts. Mechanisms involved in OTM include mechano-sensing, sterile inflammation-mediated osteoclastogenesis on the compression side and tensile force-induced osteogenesis on the tension side. Several intracellular signaling pathways and mechanosensors including the cilia and ion channels transduce mechanical force into biochemical signals that stimulate formation of osteoclasts or osteoblasts. To date, many studies were performed in vitro or using human gingival crevicular fluid samples. Thus, the use of transgenic animals is very helpful in examining a cause and effect relationship. Key cell types that participate in mediating the response to OTM include periodontal ligament fibroblasts, mesenchymal stem cells, osteoblasts, osteocytes, and osteoclasts. Intercellular signals that stimulate cellular processes needed for orthodontic tooth movement include receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-α (TNF-α), dickkopf Wnt signaling pathway inhibitor 1 (DKK1), sclerostin, transforming growth factor beta (TGF-β), and bone morphogenetic proteins (BMPs). In this review, we critically summarize the current OTM studies using transgenic animal models in order to provide mechanistic insight into the cellular events and the molecular regulation of OTM.

Animal models of gestational diabetes: characteristics and consequences to the brain and behavior of the offspring.

Gestational diabetes (GD) is the glucose intolerance that occursduring pregnancy. Mothers who develop diabetes during gestation are at increased risk of developing type 2 diabetes mellitus (T2DM) later in life, and the risk of adverse fetal and neonatal outcomes are also increased as a function of maternal hyperglycemia. Infants who are exposed to fetal hyperglycemia show an increased risk of becoming obese and developing T2DM later in life. Due to the need of new research on this field, and the difficulty of performing studies in human brain, studies using experimental models are necessary to suggest possible ways to avoid or inhibit offspring brain damage or harmful metabolic alterations. Here, it was made a review about the characteristics of the main animal models of GD, and what are the consequences to the brain and behavior of the offspring. In many experimental models, either by pharmacological induction, diet manipulation, or in the use of transgenic animals, glycemic conditions are severe. S961, a selective insulin receptor antagonist, revealed an increased fasting blood glucose level and glucose intolerance during mid-gestation, which returned to basal levels postpartum in mice. GD contributes to offspring neuroinflammation, influences neuronal distribution in central nervous system (CNS), and apoptosis during embryogenesis, which in turn may contribute to changes in behavior and memory in adult life and aging. The usage of animal models to study GD allows to examine extensively the characteristics of this condition, the molecular mechanisms involved and the consequences to the brain and behavior of the offspring.

The use of transgenic animals to enhance people and animal health

The use of transgenic animals to enhance people and animal health

Recombinant Technologies to Improve Ruminant Production Systems: The Past, Present and Future

The use of recombinant technologies has been proposed as an alternative to improve livestock production systems for more than 25 years. However, its effects on animal health and performance have not been described. Thus, understanding the use of recombinant technology could help to improve public acceptance. The objective of this review is to describe the effects of recombinant technologies and proteins on the performance, health status, and rumen fermentation of meat and milk ruminants. The heterologous expression and purification of proteins mainly include eukaryotic and prokaryotic systems like Escherichia coli and Pichia pastoris. Recombinant hormones have been commercially available since 1992, their effects remarkably improving both the reproductive and productive performance of animals. More recently the use of recombinant antigens and immune cells have proven to be effective in increasing meat and milk production in ruminant production systems. Likewise, the use of recombinant vaccines could help to reduce drug resistance developed by parasites and improve animal health. Recombinant enzymes and probiotics could help to enhance rumen fermentation and animal efficiency. Likewise, the use of recombinant technologies has been extended to the food industry as a strategy to enhance the organoleptic properties of animal-food sources, reduce food waste and mitigate the environmental impact. Despite these promising results, many of these recombinant technologies are still highly experimental. Thus, the feasibility of these technologies should be carefully addressed before implementation. Alternatively, the use of transgenic animals and the development of genome editing technology has expanded the frontiers in science and research. However, their use and implementation depend on complex policies and regulations that are still under development.

Comparison of chloroquine-like molecules for lysosomal inhibition and measurement of autophagic flux in the brain

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.

Application of Genetically Engineered Pigs in Biomedical Research.

Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. Animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. Another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. The further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. Numerous studies have shown that the pig (Sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. Short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species This review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models.

Current fluid biomarkers, animal models, and imaging tools for diagnosing chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder that results from repetitive traumatic brain injury (TBI), whether mild or severe. Several popular sports that subject the head to impact have been linked as a primary cause of the disease. Phosphorylated tau and Aβ deposits are the two proteins observed histopathologically in CTE patients. An ischemic environment is created that contributes to the hyperphosphorylation of tau following traumatic brain injury. The use of fluid biomarkers, animal models for TBI, as well as imaging tools are considered valuable in understanding the pathophysiological mechanism of CTE. This review gives particular attention to the characteristics, advantages, and disadvantages of the current fluid biomarkers, use of animal models, and imaging techniques used in CTE diagnosis. Beta-amyloid and phosphorylated tau were suggested as the two main pathological biomarkers for chronic traumatic encephalopathy (CTE) diagnosis, though research into other fluid biomarkers of traumatic brain injury (TBI) such as neurofilament light chain (NFL), glial fibrillary acidic protein (GFAP), and C-C motif chemokine 11 (CCL11) has been undertaken but was mostly limited by sample size, and decreased sensitivity in follow-up studies. Animal models and devices that simulate TBI were valuable in exploring injury dynamics and the role it may have on CTE. The use of transgenic animals in CTE research has also uncovered the different risk genes that may enhance CTE pathology. Magnetic resonance imaging (MRI), functional MRI and positron emission tomography (PET) imaging showed enough resolution to accurately diagnose CTE. However, diffusion tensor imaging (DTI) was able to identify microstructural changes in professional boxers that were not apparent in MRI. Currently, a single biomarker or imaging technique is not enough to accurately diagnose CTE and diagnostic accuracy is significantly enhanced when these different parameters are combined.

THU-376-A viral mediated liver repopulation assay without the use of transgenic animals

THU-376-A viral mediated liver repopulation assay without the use of transgenic animals

Production of Recombinant Proteins in the Milk of Transgenic Animals: Current State and Prospects

The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk. The current state and prospects for the development of this area of biotechnology are discussed in relation to the emergence of novel genome editing technologies. Experimental and practical techniques are briefly discussed.

Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development

SUMMARYA standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.

Моделирование болезни Альцгеймера на животных

Alzheimer’s disease is a common neurodegenerative disease characterized by memory impairment and a progressive incurable cognitive decline. Modeling Alzheimer’s disease in animals allows to study the pathogenesis of the disease, to conduct preclinical studies, to search for possible ways to stop the development and progression of the disease. Since the etiology of Alzheimer’s disease remains unknown, there is no «natural» biological model that fully reflects the set of pathological disorders that occur in a person with this pathology, and among a variety of options of pathogenetic models there is no generally accepted one. This review provides an analysis of data on modern approaches to modeling Alzheimer’s disease in animals. The important aspects to be taken into account in modeling (metabolic disorders of β-amyloid and tau protein, the role of inflammatory reactions, etc.) have been analyzed. We review such widespread models as the use of transgenic animals of the frst and second generations expressing human genes with mutations found in the family form of the disease; models of natural aging and senescence-accelerated animals; intraventricular neurotoxin injection models. We also discuss more rare surgical models that phenotypically simulate some aspects of Alzheimer’s disease. An important role of inflammatory reactions in the pathogenesis of the disease is analyzed, which causes interest in models of neuroinflammation that manifests itself before the development of other pathological disorders characteristic for Alzheimer’s disease. We assess the advantages and limitations of each model, the features of their application, the main results obtained with their help, promising directions for further researches.

Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction.

Synapses that are overproduced during histogenesis in the nervous system are eventually lost and connectivity is refined. Membrane receptor signaling leads to activity-dependent mutual influence and competition between axons directly or with the involvement of the postsynaptic cell and the associated glial cell/s. Presynaptic muscarinic acetylcholine (ACh) receptors (subtypes mAChR; M1, M2 and M4), adenosine receptors (AR; A1 and A2A) and the tropomyosin-related kinase B receptor (TrkB), among others, all cooperate in synapse elimination. Between these receptors there are several synergistic, antagonic and modulatory relations that clearly affect synapse elimination. Metabotropic receptors converge in a limited repertoire of intracellular effector kinases, particularly serine protein kinases A and C (PKA and PKC), to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. In most cells A1, M1 and TrkB operate mainly by stimulating PKC whereas A2A, M2 and M4 inhibit PKA. We hypothesize that a membrane receptor-induced shifting in the protein kinases A and C activity (inhibition of PKA and/or stimulation of PKC) in some nerve endings may play an important role in promoting developmental synapse elimination at the neuromuscular junction (NMJ). This hypothesis is supported by: (i) the tonic effect (shown by using selective inhibitors) of several membrane receptors that accelerates axon loss between postnatal days P5–P9; (ii) the synergistic, antagonic and modulatory effects (shown by paired inhibition) of the receptors on axonal loss; (iii) the fact that the coupling of these receptors activates/inhibits the intracellular serine kinases; and (iv) the increase of the PKA activity, the reduction of the PKC activity or, in most cases, both situations simultaneously that presumably occurs in all the situations of singly and paired inhibition of the mAChR, AR and TrkB receptors. The use of transgenic animals and various combinations of selective and specific PKA and PKC inhibitors could help to elucidate the role of these kinases in synapse maturation.

Inhibition of Inflammation Mediated Through the Tumor Necrosis Factor α Biochemical Pathway Can Lead to Favorable Outcomes in Alzheimer Disease.

Tumor necrosis factor α (TNF-α) inhibitors have long been used as disease-modifying agents in immune disorders. Recently, research has shown a role of chronic neuroinflammation in the pathophysiology of neurodegenerative diseases such as Alzheimer disease, and interest has been generated in the use of anti-TNF agents and TNF-modulating agents for prevention and treatment. This article extensively reviewed literature on animal studies testing these agents. The results showed a role for direct and indirect TNF-α inhibition through agents such as thalidomide, 3,6-dithiothalidomide, etanercept, infliximab, exendin-4, sodium hydrosulfide, minocycline, imipramine, and atorvastatin. Studies were performed on mice, rats, and monkeys, with induction of neurodegenerative physiology either through the use of chemical agents or through the use of transgenic animals. Most of these agents showed an improvement in cognitive function as tested with the Morris water maze, and immunohistochemical and histopathological staining studies consistently showed better outcomes with these agents. Brains of treated animals showed significant reduction in pro-inflammatory TNF-α and reduced the burden of neurofibrillary tangles, amyloid precursor protein, and β-amyloid plaques. Also, recruitment of microglial cells in the central nervous system was significantly reduced through these drugs. These studies provide a clearer mechanistic understanding of the role of TNF-α modulation in Alzheimer disease. All studies in this review explored the use of these drugs as prophylactic agents to prevent Alzheimer disease through immune modulation of the TNF inflammatory pathway, and their success highlights the need for further research of these drugs as therapeutic agents.

Research and development on lactoferrin and its derivatives in China from 2011-2015.

Lactoferrin (Lf), a multifunctional glycoprotein, is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. Lactoferricin (Lfcin) is located in the N-terminal region of this protein. In this review, the current state of research into Lf and Lfcin in China is described. Searching with HistCite software in Web Sci located 118 papers published by Chinese researchers from 2011-2015, making China one of the top 3 producers of Lf research and development in the world. The biological functions of Lf and Lfcin are discussed, including antibacterial, antiviral, antifungal, anticarcinogenic, and anti-inflammatory activities; targeted drug delivery, induction of neurocyte, osteoblast, and tenocyte growth, and possible mechanisms of action. The preparation and heterologous expression of Lf in animals, bacteria, and yeast are discussed in detail. Five Lf-related food additive factories and 9 Lf-related health food production companies are certified by the China Food and Drug Administration (CFDA). The latest progress in the generation of transgenic livestock in China, the safety of the use of transgenic animals, and future prospects for the uses of Lf and Lfcin are also covered.