Advances In Transgenic Technology Research Articles

Transgenic Improvement for Biotic Resistance of Crops.

Biotic constraints, including pathogenic fungi, viruses and bacteria, herbivory insects, as well as parasitic nematodes, cause significant yield loss and quality deterioration of crops. The effect of conventional management of these biotic constraints is limited. The advances in transgenic technologies provide a direct and directional approach to improve crops for biotic resistance. More than a hundred transgenic events and hundreds of cultivars resistant to herbivory insects, pathogenic viruses, and fungi have been developed by the heterologous expression of exogenous genes and RNAi, authorized for cultivation and market, and resulted in a significant reduction in yield loss and quality deterioration. However, the exploration of transgenic improvement for resistance to bacteria and nematodes by overexpression of endogenous genes and RNAi remains at the testing stage. Recent advances in RNAi and CRISPR/Cas technologies open up possibilities to improve the resistance of crops to pathogenic bacteria and plant parasitic nematodes, as well as other biotic constraints.

Advances and Perspectives of Transgenic Technology and Biotechnological Application in Forest Trees.

Transgenic technology is increasingly used in forest-tree breeding to overcome the disadvantages of traditional breeding methods, such as a long breeding cycle, complex cultivation environment, and complicated procedures. By introducing exogenous DNA, genes tightly related or contributed to ideal traits—including insect, disease, and herbicide resistance—were transferred into diverse forest trees, and genetically modified (GM) trees including poplars were cultivated. It is beneficial to develop new varieties of GM trees of high quality and promote the genetic improvement of forests. However, the low transformation efficiency has hampered the cultivation of GM trees and the identification of the molecular genetic mechanism in forest trees compared to annual herbaceous plants such as Oryza sativa. In this study, we reviewed advances in transgenic technology of forest trees, including the principles, advantages and disadvantages of diverse genetic transformation methods, and their application for trait improvement. The review provides insight into the establishment and improvement of genetic transformation systems for forest tree species. Challenges and perspectives pertaining to the genetic transformation of forest trees are also discussed.

Seed Mucilage: Biological Functions and Potential Applications in Biotechnology.

In plants, the diaspore (seed dispersal unit) may include a seed coat and/or pericarp to protect the embryo and assist in dispersion. In many species, the seed coat and/or pericarp secrete a gelatinous mixture of cell wall polysaccharides known as mucilage. In several species, mucilage synthesis, secretion and modification have been studied extensively as model systems for the investigation of plant cell wall structure and function. Despite this, efforts toward understanding the role of mucilage have received less attention. Mucilage has been hypothesized to impact seed dispersal through interaction with soil, protecting the seed in the gut following ingestion by animals or affecting the ability of seeds to sink or float in water. Mucilage has been found to influence seed germination and seedling establishment, most often during abiotic stress, probably by maintaining seed hydration when water is scarce. Finally, mucilage has been documented to mediate interactions with various organisms. Advances in transgenic technology should enable the genetic modification of mucilage structure and function in crop plants. Cells synthesizing mucilage may also be a suitable platform for creating custom polysaccharides or proteins with industrial applications. Thus, in the near future, it is likely that research on seed mucilage will expand well beyond the current focus. Here we summarize our understanding of the biological functions of mucilage and provide an outlook on the future of mucilage research.

Animal Models for Parkinson's Disease Research: Trends in the 2000s.

Parkinson’s disease (PD) is a chronic and progressive movement disorder and the second most common neurodegenerative disease. Although many studies have been conducted, there is an unmet clinical need to develop new treatments because, currently, only symptomatic therapies are available. To achieve this goal, clarification of the pathology is required. Attempts have been made to emulate human PD and various animal models have been developed over the decades. Neurotoxin models have been commonly used for PD research. Recently, advances in transgenic technology have enabled the development of genetic models that help to identify new approaches in PD research. However, PD animal model trends have not been investigated. Revealing the trends for PD research will be valuable for increasing our understanding of the positive and negative aspects of each model. In this article, we clarified the trends for animal models that were used to research PD in the 2000s, and we discussed each model based on these trends.

High-resolution transcriptional profiling of Anopheles gambiae spermatogenesis reveals mechanisms of sex chromosome regulation

Although of high priority for the development of genetic tools to control malaria-transmitting mosquitoes, only a few germline-specific regulatory regions have been characterised to date and the presence of global regulatory mechanisms, such as dosage compensation and meiotic sex chromosome inactivation (MSCI), are mostly assumed from transcriptomic analyses of reproductive tissues or whole gonads. In such studies, samples include a significant portion of somatic tissues inevitably complicating the reconstruction of a defined transcriptional map of gametogenesis. By exploiting recent advances in transgenic technologies and gene editing tools, combined with fluorescence-activated cell sorting and RNA sequencing, we have separated four distinct cell lineages from the Anopheles gambiae male gonads: premeiotic, meiotic (primary and secondary spermatocytes) and postmeiotic. By comparing the overall expression levels of X-linked and autosomal genes across the four populations, we revealed a striking transcriptional repression of the X chromosome coincident with the meiotic phase, classifiable as MSCI, and highlighted genes that may evade silencing. In addition, chromosome-wide median expression ratios of the premeiotic population confirmed the absence of dosage compensation in the male germline. Applying differential expression analysis, we highlighted genes and transcript isoforms enriched at specific timepoints and reconstructed the expression dynamics of the main biological processes regulating the key stages of sperm development and maturation. We generated the first transcriptomic atlas of A. gambiae spermatogenesis that will expand the available toolbox for the genetic engineering of vector control technologies. We also describe an innovative and multidimensional approach to isolate specific cell lineages that can be used for the targeted analysis of other A. gambiae organs or transferred to other medically relevant species and model organisms.

Modeling phenotypes of malignant gliomas.

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single‐cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.

Abscisic acid and abiotic stress tolerance – Different tiers of regulation

Abiotic stresses affect plant growth, metabolism and sustainability in a significant way and hinder plant productivity. Plants combat these stresses in myriad ways. The analysis of the mechanisms underlying abiotic stress tolerance has led to the identification of a highly complex, yet tightly regulated signal transduction pathway consisting of phosphatases, kinases, transcription factors and other regulatory elements. It is becoming increasingly clear that also epigenetic processes cooperate in a concerted manner with ABA-mediated gene expression in combating stress conditions. Dynamic stress-induced mechanisms, involving changes in the apoplastic pool of ABA, are transmitted by a chain of phosphatases and kinases, resulting in the expression of stress inducible genes. Processes involving DNA methylation and chromatin modification as well as post transcriptional, post translational and epigenetic control mechanisms, forming multiple tiers of regulation, regulate this gene expression. With recent advances in transgenic technology, it has now become possible to engineer plants expressing stress-inducible genes under the control of an inducible promoter, enhancing their ability to withstand adverse conditions. This review briefly discusses the synthesis of ABA, components of the ABA signal transduction pathway and the plants' responses at the genetic and epigenetic levels. It further focuses on the role of RNAs in regulating stress responses and various approaches to develop stress-tolerant transgenic plants.

Prion Disease: A Tale of Folds and Strains

Research on prions, the infectious agents of devastating neurological diseases in humans and animals, has been in the forefront of developing the concept of protein aggregation diseases. Prion diseases are distinguished from other neurodegenerative diseases by three peculiarities. First, prion diseases, in addition to being sporadic or genetic like all other neurodegenerative diseases, are infectious diseases. Animal models were developed early on (a long time before the advent of transgenic technology), and this has made possible the discovery of the prion protein as the infectious agent. Second, human prion diseases have true equivalents in animals, such as scrapie, which has been the subject of experimental research for many years. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonosis caused by bovine spongiform encephalopathy (BSE) prions. Third, they show a wide variety of phenotypes in humans and animals, much wider than the variants of any other sporadic or genetic neurodegenerative disease. It has now become firmly established that particular PrP(Sc) isoforms are closely related to specific human prion strains. The variety of human prion diseases, still an enigma in its own right, is a focus of this article. Recently, a series of experiments has shown that the concept of aberrant protein folding and templating, first developed for prions, may apply to a variety of neurodegenerative diseases. In the wake of these discoveries, the term prion has come to be used for Aβ, α-synuclein, tau and possibly others. The self-propagation of alternative conformations seems to be the common denominator of these "prions," which in future, in order to avoid confusion, may have to be specified either as "neurodegenerative prions" or "infectious prions."

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10 μg/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.

Food security: Overcome India's GM crops prejudice

India's agricultural productivity is not keeping pace with its population, despite its growing economy. Advances in transgenic technology could alleviate the situation, but there is strident opposition in the nation to genetically modified (GM) crops from non-governmental organizations and from spiritual gurus, in the name of tradition, biodiversity and other ill-founded concerns. Millions of people worldwide have been consuming GM maize (known as Bt corn) over the past decade, and no authenticated health issues have arisen. Bt-transformed plants reduce pesticide application so are suitable for organic farming. GM rice that provides a balance of protein and micronutrients would vastly improve the health of millions of children.

Meeting Report of the National Symposium on “Advances in Transgenic Technology and Its Impact on the National Economy” held 23rd April 2010 at Allahabad, UP, India

The National Symposium on ‘‘Advances in Transgenic Technology and its Impact on National Economy’’, stressed the importance of transgenic technology for overcoming the growing problem of food scarcity. The intention was for participants to consider the application of transgenic technology outside their own specific field of research, and examine the different attitudes, demands, questions and challenges of transgenic development in India. Besides highlighting the newest scientific developments the presentations focused on commercial and legal aspects of transgenic technology. The controversy attached to genetically engineered food and cloned organisms has led to much discussion within the scientific community and the public on how these advances impact the food supply, Medicare, legal system, our economy—our daily lives. Although transgenic research will have a bearing on many disciplines, forums where a diverse range of experts from the various stakeholder communities are rare and across these disciples there is limited exchange of their research results, ideas or questions. The brain storming symposium was opened by the Honourable Vice Chancellor (RBL co-author) of the host venue SHIATS and set the stage by describing animal biotechnology as an ancient human activity. The first session started with a talk by Ashok Pandey (National Institute for Interdisciplinary Science and Technology, CSIR) on the topic ‘‘GE crops: Status and Perspectives—What is good for India’’. He highlighted the relevance of biotechnological tools to meet the demands of the escalating world population. Based on the statistics of the WHO, by the year 2050 there should be a three times increase in food productivity to feed the growing population. The global agricultural growth had decreased from 3% in 1960 to 2% in 2003. Even though conventional breeding and good farm practices have been employed for increasing crop productivity it is still unable to meet the current food demand. The increasing population of the country has to rely on a small percentage of farmland for its food supply, a major problem that will increase in the future. Pandey deliberated on a range of significant issues such as, How to produce and store the quantity of food the world requires today and would require in near future?; the potential benefits of genetically engineered (GE) crops; Social and environmental issues related with GE crops and; the status of GE crop grown in India. Emphasized that even with good M. Devasahayam E. P. Koshy V. K. Morya Jacob School of Biotechnology and Bioengineering, SHIATS, Allahabad, UP 211007, India

High field BOLD response to forepaw stimulation in the mouse

We have established a robust protocol for longitudinal fMRI in mice at high field MRI using a medetomidine anesthesia. Electrical forepaw stimulation in anesthetized animals is widely used to produce BOLD contrast in the primary somatosensory cortex. To preserve neuronal activity, most fMRI experiments used α-chloralose to produce sedation, but severe side effects make this procedure unsuitable for survival experiments. As advantageous alternative, the α2-adrenergic receptor agonist medetomidine has been applied successfully to permit longitudinal fMRI studies in rats. With the advent of transgenic technology, mouse models have become increasingly attractive raising the demand for implementation of a suitable fMRI protocol for mice. Therefore, we investigated the use of medetomidine for repetitive fMRI experiments in C57BL/6 mice. We evaluated the optimal medetomidine dose for subcutaneous application. Somatosensory evoked potentials (SSEPs) in the contralateral somatosensory cortex were recorded to assess brain activity under medetominidine following forepaw stimulation. Repetitive administration of medetomidine, the requirement for longitudinal brain activation studies, was well tolerated. Using the forepaw stimulation paradigm, we observed BOLD contrast in the contralateral somatosensory cortex in ∼50% of the performed scans using gradient echo-echo planar imaging (GE-EPI). However, imaging the small mouse brain at high field strength is challenging and we observed strong susceptibility artifacts in GE-EPI images in the cortex. We have developed an agar gel cap for successful compensation of these artifacts as prerequisite for successful mouse fMRI at 11.7T. The established protocol will be suitable for brain activation studies in transgenic animals and for studies of functional deficit and recovery after brain injury in mice.

Profile for Amyloid-β and Tau Expression in Primary Cortical Cultures from 3xTg-AD Mice

Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 (M146V), APP (Swe) and tau (P301L) human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca(2+)] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-beta and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.

Advances in the generation of mouse models to elucidate the pathways of drug metabolism in rodents and man

Animal models remain a cornerstone of drug development; however, such studies have a major limitation in that pathways of drug metabolism can vary markedly from those in humans, resulting in profound species differences in drug efficacy, tox-icity and pharmacokinetics. Advances in transgenic technology have now allowed the generation of animal models, in which endogenous enzymes are replaced with their human counterparts, thereby creat-ing a system that more closely reflects the human situation. In this article, we will describe advances in this research area, focusing on the strengths and limitations of recent mouse models that will undoubt-edly assist in overcoming some of the issues associated with species-specific differences in drug-metabolism studies.An adaptive response system has evolved in mammalian species, which protects them against a chemically challenging environment. This detoxification system consists of a complex network of xeno-receptors, drug-metabolizing enzymes (DMEs) and transporters, and it does not differentiate between synthetic chemicals, drugs or environmental toxins. Although transporters undoubtedly play an impor-tant role in drug disposition, they will not be discussed further in this article and the reader is referred to recent publica-tions

Simultaneous behavioral characterizations: Embracing complexity

The advent of transgenic technologies in mice has dramatically impacted the genetic analysis of behavior. The first knockout mice evaluated for behavioral responses were reported in 1992 (1, 2), and since these initial reports a significant number of scientists have performed behavioral studies with mutant mice each year. In fact, a literature search by using simply the words “knockout” and “behavior” and “mice” results in almost 4,000 hits. The powerful molecular technique has not simply increased the amount of behavioral genetic studies performed across the globe, but has fundamentally altered the strategies and methodologies of standard approaches for assessing rodent behavior. Not only have standard experimental designs been altered to successfully work with transgenic mice, but scientists have also developed technologies to simultaneously and automatically quantify fine behavioral patterns in home cage settings, as illustrated by a study in this issue of PNAS (3). This study clearly demonstrates the capacity to capture and characterize complex behavior in mice coupled with sophisticated learning algorithms to provide a type of detailed analysis that has not been previously available.

Transgenic Leishmania and the immune response to infection.

Genetic manipulation of single-celled organisms such as the Leishmania parasite enables in depth analysis of the consequences of genotypic change on biological function. In probing the immune responses to infection, use of transgenic Leishmania has the potential to unravel both the contribution of the parasite to the infection process and the cellular interactions and mechanisms that characterize the innate and adaptive immune responses of the host. Here, we briefly review recent technical advances in parasite genetics and explore how these methods are being used to investigate parasite virulence factors, elucidate immune regulatory mechanisms and contribute to the development of novel therapeutics for the leishmaniases. Recent developments in imaging technology, such as bioluminescence and intravital imaging, combined with parasite transfection with fluorescent or enzyme-encoding marker genes, provides a rich opportunity for novel assessment of intimate, real-time host-parasite interactions at a previously unexplored level. Further advances in transgenic technology, such as the introduction of robust inducible gene cassettes for expression in intracellular parasite stages or the development of RNA interference methods for down-regulation of parasite gene expression in the host, will further advance our ability to probe host-parasite interactions and unravel disease-promoting mechanisms in the leishmaniases.

Animal models of asthma

Animal models of asthma are a tool that allows studies to be conducted in the setting of an intact immune and respiratory system. These models have highlighted the importance of T-helper type 2 driven allergic responses in the progression of asthma and have been useful in the identification of potential drug targets for interventions involving allergic pathways. However, a number of drugs that have been shown to have some efficacy in animal models of asthma have shown little clinical benefit in human asthmatics. This may be due to a number of factors including the species of animal chosen and the methods used to induce an asthmatic phenotype in animals that do not normally develop a disease that could be characterized as asthma. The range of animal models available is vast, with the most popular models being rodents (inbred mice and rats) and guinea-pigs, which have the benefit of being easy to handle and being relatively cost effective compared with other models that are available. The recent advances in transgenic technology and the development of species-specific probes, particularly in mice, have allowed detailed mechanistic studies to be conducted. Despite these advances in technology, there are a number of issues with current animal models of asthma that must be recognized including the disparity in immunology and anatomy between these species and humans, the requirement for adjuvant during senitization in most models, the acute nature of the allergic response that is induced and the use of adult animals as the primary disease model. Some larger animal models using sheep and dogs have been developed that may address some of these issues but they also have different biology from humans in many ways and are extremely costly, with very few probes available for characterizing allergic responses in the airway in these species. As research in this area continues to expand, the relative merits and limitations of each model must be defined and understood in order to evaluate the information that is obtained from these models and to extrapolate these findings to humans so that effective drug therapies can be developed. Despite these issues, animal models have been, and will continue to be, vital in understanding the mechanisms that are involved in the development and progression of asthma.

Defining the Gonadotrophin‐Releasing Hormone Neuronal Network: Transgenic Approaches to Understanding Neurocircuitry

The gonadotrophin-releasing hormone (GnRH) neurones are the final downstream effector neurones driving the central regulation of reproductive function and fertility in all mammalian species. Although it is abundantly clear that successful fertility relies upon the communication of a variety signals regarding internal and external cues to the GnRH neuronal population, how this is achieved remains poorly understood. A range of technical limitations has posed significant hurdles to defining, with any certainty, the complexities of the synaptic neuronal network regulating GnRH neurones. However, recent advances in transgenic technology have opened up new avenues to reconsider questions aimed at understanding this critical network. This article addresses some of the latest advances that use transgenic mouse models as tools to understand the neuronal circuitry underpinning the regulation of the GnRH neurones. By incorporating standard morphological and viral tract tracing techniques with innovative transgenic tools, recent studies have uncovered previously unappreciated qualities of the GnRH neurone, including extensive dendritic lengths, numerous somal and dendritic spines and plasticity over pubertal development, along with beginning to define the primary and higher-order afferents that make up the GnRH neuronal network.

Microbial community structure in soils with decomposing residues from plants with genetic modifications to lignin biosynthesis

Lignin is a major determinant of the decomposition of plant materials in soils. Advances in transgenic technology have led to the possibility of modifying lignin to improve the pulping properties of plant materials for papermaking. Previous studies have shown that lignin modifications also affect the rate of plant material decay in soil. The aim of this work was to investigate short-term changes in soil microbial community structures when tobacco residues with reduced activity of enzymes in the monolignol pathway decompose. The residues from lignin-modified plants all decomposed faster than unmodified plant materials. The relative proportions of some of the structural groups of microbial phospholipid fatty acids were affected by genetic modifications, especially the proportion of double unsaturated chain fatty acids, indicative of fungi.

Use of Transgenic Mice to Study Lung Morphogenesis and Function

Successful transition to air breathing at birth depends on perinatal maturation of the gas exchange surface, resorption of fluid from the air spaces, and synthesis and secretion of pulmonary surfactant. Genetic mutations that alter lung development and/or cellular differentiation in the prenatal period, lung function in the perinatal period, or lung homeostasis in the postnatal period can lead to neonatal lethality or chronic lung disease. Current knowledge of the molecular pathways that regulate key prenatal, perinatal, and postnatal morphogenetic events has been shaped largely by remarkable advances in transgenic technologies. In this review, selected transgenic mouse models are highlighted to illustrate the power of this technology, which in many cases has provided important insights that otherwise could not have been obtained.