Development Of Transgenic Technology Research Articles

Analysis of Genetically Modified Food Induced International Trade Law Issues

The advantages of genetically modified food are gradually highlighted because of the rapid development of modern transgenic technology. The trade proportion of genetically modified food in international trade products is increasing. Till now, legal regulations concerning the international trade of genetically modified food are still contradictory and conflicting, and a series of initiated legal issues about the international trade of genetically modified food becomes more acute. As a great power of transgenic crops planting, China should fully learn from the trade disputes of genetically modified food, perfect laws such as the safety management, approval and identification of import and export, accelerate the development of transgenic technology, and strengthen the mutual benefit and collaboration of the developing countries, thus to gain a strong competitive position in international trade and maintain the fundamental interests of China better.

Innovations in genetically modified agricultural technologies in China’s public sector

PurposeThe purpose of this paper is to assess whether China’s public sector can continue to generate advanced genetically modified (GM) technologies that will be competitive in the market.Design/methodology/approachThe authors investigated all the research teams that have been conducting research projects under the variety development special program. The data collected include detail information on research capacity, research areas, performance, and process of their research projects. Based on the survey data, the authors assessed the innovations and progress of the variety development special program.FindingsUnlike other countries, most GM products in China are developed by public research institutes. There is rising concern on the ability of China’s public sector to continuously generate indigenous GM technology that can compete with multinational companies. The study surveyed 197 research institutes and 487 research teams and found that the GM program in China lacks coordination: researchers do not want to share their research materials with others. Due to the lack of coordination, most of the hundreds of research teams often worked independently in the year 2008-2010. Moreover, the authors found the lack of coordination may be due to the reason that the interests of researchers are not well protected. This paper also provided the recent progress and policy changes of GM program in China, and it found that the efficiency in the later three years improved a lot. In order to establish a competitive national public GM research system, China should continuously consolidate and integrate the upstream, midstream, and downstream activities of the whole GM innovation process. China’s public sector may also need to work more closely with both the domestic and international private sectors.Originality/valueThis paper is a comprehensive analysis on the development of transgenic technology in China. The results of this paper can provide evidence for the dynamic adjustment of the policies in the variety development special program and can also provide reference for the future assessment of the variety development special program.

Generation of a transgenic cashmere goat using the piggyBac transposition system

The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT). The embryos (n = 40) were implanted into female goats (n = 20). One transgenic kid that expressed EGFP throughout the surface features of its body was born. This result demonstrated the usefulness of PB transposon system in generating transgenic Cashmere goats.

Expression of Cucumber Green Mottle Mosaic Virus Movement Protein in Cucumber Leads to the Expression Changes of Endogenous Gene

Cucumber green mottle mosaic virus (CGMMV) is one of the most important diseases of cucurbit crops. To date the only method available to control this devastating disease is the use of resistant varieties or disease-resistant rootstocks. However, the development of transgenic technology offers the potential to create resistant varieties through the expression of foreign genes. Such approaches are not without risk, and it has been noted that introduction of transgenes can have wide ranging effects, often affecting non-target processes. The current study was therefore initiated to investigate the effect of genetic modification on 12 related genes in transgenic cucumber seedlings expressing the CGMMV movement protein (CGMMV-MP) at the two-true-leaf stage. Compared with non-transgenic cucumbers (cv. Zhongnong 16), the results of quantitative PCR (qPCR) indicated that six of the genes had significant altered expression in the transgenic plants, four that were up-regulated including the cucumber peeling cupredoxin, Histone H4, Cytochrome oxidase and Thaumatin-like protein and two that were down-regulated, cytochrome b6-f complex and disulfide isomerase. The data collected therefore provide greater understanding of the impact of introduced exogenous genes in cucumber, as well as highlighting resistance genes that have the potential to prevent CGMMV infection.

Optimal barrier zones for stopping the invasion of Aedes aegypti mosquitoes via transgenic or sterile insect techniques

Biological invasions have dramatically altered the natural world by threatening native species and their communities. Moreover, when the invading species is a vector for human disease, there are further substantive public health and economic impacts. The development of transgenic technologies is being explored in relation to new approaches for the biological control of insect pests. We investigate the use of two control strategies, classical sterile insect techniques and transgenic late-acting bisex lethality (Release of Insects carrying a Dominant Lethal), for controlling invasion of the mosquito Aedes aegypti using a spatial stage-structured mathematical model. In particular, we explore the use of a barrier zone of sterile/transgenic insects to prevent or impede the invasion of mosquitoes. We show that the level of control required is not only highly sensitive to the rate at which the sterile/transgenic males are released in the barrier zone but also to the spatial range of release. Our models characterise how the distribution of sterile/transgenic mosquitoes in the barrier zone can be controlled so as to minimise the number of mass-produced insects required for the arrest of species invasion. We predict that, given unknown rates of mosquito dispersal, management strategies should concentrate on larger release areas rather than more intense release rates for optimal control.

337 PRODUCTION OF TRANSGENIC SHEEP USING RECOMBINANT LENTIVIRUS MICROINJECTION OF IN VIVO PRODUCED EMBRYOS

The development of transgenic technology has enormous potential for livestock improvement. Several methods have been employed for generating transgenic livestock to date, the most popular being pronuclear injection or somatic cell nuclear transfer using genetically modified cells. However, the high cost and relatively low efficiencies seen with these methods have prompted development of alternative methods for producing transgenic livestock. Recently, the introduction of transgenes using viral vectors, in particular lentiviral vectors, has provided an avenue for increasing the efficiency of transgenic livestock production. Microinjection of concentrated lentivirus into the perivitelline space of oocytes and zygotes has been demonstrated as an effective means of creating transgenic livestock (cattle, sheep, and swine). In this study, we investigated the efficiency of producing transgenic sheep through microinjection of recombinant lentivirus into in vivo produced zygotes. Recombinant lentivirus was produced through co-transfection of HEK293T cells with various lentiviral transfer plasmids (each coding for a short hairpin RNA and a fluorescent marker protein) as well as a packaging plasmid and a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), which was used to pseudotype viral particles. For lentivirus concentration, a total of 36 mL of viral supernatant was produced per viral construct. Viral supernatant was ultracentrifuged on a 15% sucrose cushion at 4°C for 1.5 h at a speed of 50 000 × g. The resulting viral pellet was resuspended in 30 μL of PBS and virus was frozen at –80°C until needed. Zygotes were surgically flushed from oviducts of superovulated donor ewes 24 h post-mating. Concentrated recombinant lentivirus (titer ≥ IU mL–1) was then microinjected into the perivitelline space. Injected embryos were surgically transferred into the oviduct of synchronized recipient ewes immediately after injection (3–4 embryos per ewe). Pregnancies were confirmed by ultrasound at 35 days of gestation. The pregnancy rate was 39% (45/114 ewes exhibiting at least one viable fetus). A subset of pregnant ewes was killed at 60 to 70 days gestation to harvest a total of 36 fetuses. Analysis of transgene incorporation was performed by PCR using genomic DNA isolated from skin and liver tissue samples. Two independent PCR reactions were performed per sample, and PCR analysis revealed 14 of the collected fetuses to be transgenic (39%). The remaining pregnancies were allowed to progress to term, and 32 lambs were born. Genomic DNA was isolated from blood samples collected on each lamb, and PCR analysis was performed as above. Of the lambs born, 13 of 32 (40%) were confirmed to be transgenic by PCR analysis. Southern blot analysis is currently underway to confirm PCR data. These results demonstrate that microinjection of recombinant lentivirus into in vivo produced sheep zygotes is an effective and efficient method for generating transgenic sheep.

Transgenic rhesus monkeys produced by gene transfer into early-cleavage–stage embryos using a simian immunodeficiency virus-based vector

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage-stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.

Modelling pulsed releases for sterile insect techniques: fitness costs of sterile and transgenic males and the effects on mosquito dynamics

Summary 1. The development of transgenic technologies, coupled with sterile insect techniques (SIT), is being explored in relation to new approaches for the biological control of insect pests. Recent studies have shown that there are often fitness costs associated with transgenic insect strains, but the impact of these costs on their potential use in pest control is poorly understood. 2. In this paper, we explore the impact of an insect fitness cost on two control strategies (classical SIT and transgenic late‐acting bisex lethality) using a stage‐structured mathematical model, which is parameterized for the mosquito Aedes aegypti. Counter to the majority of studies, we use realistic pulsed release strategies and incorporate a fitness cost, which is manifested as a reduction in male mating competitiveness. 3. For both models we show that the level of control of a pest mosquito population is highly sensitive to the rate at which the transgenic or sterile males are released. Population control is more effective when smaller numbers of sterile/transgenic males are released more frequently than larger and less frequent releases. 4. If the wild‐type mosquito population exhibits cycles of peaks and troughs in abundance, as is the case for many insect species, then high frequency releases of transgenic males not only reduce mosquito abundance, but they may dampen future pest outbreaks, whereas the use of SIT alone may have an adverse effect, causing an increase in mosquito abundance. Additionally, the timing of sterile/transgenic male release during the mosquito population cycle is critical in reducing pest outbreak levels. 5. In all cases, the reduced fitness of the sterile/transgenic males causes reductions in control, thus requiring more frequent or greater magnitude releases. 6. Synthesis and applications. The sterile insect technique is considered to be a valuable non‐chemical tool for pest management. With the potential application of recent genetic developments to enhance the technique, it is becoming increasingly important to consider the wider ecological implications of this biological control strategy. Predicting the most efficient release strategies will be important in combating pest and vector insects as well as for limiting potential broader ecological effects. Although the focus of our models are based on the mosquito, A. aegypti, which can spread yellow fever, dengue fever and Chikungunya disease, our modelling approach and results can be applied more broadly to other species.

Fertility comparison between wild type and transgenic mice by in vitro fertilization

Transgenic mice are increasingly used as animal models for studies of gene function and regulation of mammalian genes. Although there has been continuous and remarkable progress in the development of transgenic technology over several decades, many aspects of the resulting transgenic model's phenotype cannot be completely predicted. For example, it is well known that as a consequence of the random insertion of the injected DNA construct, several founder mice of the new line need to be analyzed for possible differences in phenotype secondary to different insertion sites. The Knock out technique for transgenic production disrupts a specific gene by insertion or homologous recombination creating a null expression or replacement of the gene with a marker to localize it expression. This modification could result in pleiotropic phenotype if the gene is also expressed in tissues other than the target organs. Although the future breeding performance of the newly created model is critical to many studies, it is rarely anticipated that the new integrations could modify the reproductive profile of the new transgenic line. To date, few studies have demonstrated the difference between the parent strain's reproductive performance and the newly developed transgenic model. This study was designed to determine whether a genetic modification, knock out (KO) or transgenics, not anticipated to affect reproductive performance could affect the resulting reproductive profile of the newly developed transgenic mouse. More specifically, this study is designed to study the impact of the genetic modification on the ability of gametes to be fertilized in vitro. We analyzed the reproductive performance of mice with different background strains: FVB/N, C57BL/6 (129Sv/J x C57Bl/6)F1 and outbred CD1((R)) and compared them to mice of the same strain carrying a transgene or KO which was not anticipated to affect fertility. In vitro Fertilization was used to analyze the fertility of the mice. Oocytes from superovulated females were inseminated with sperm of same background. Fertility rate was considered as the percentage of two cell embryos scored 24 h after insemination. The data collected from this study shows that the fertilization rate is affected (reduced to half fold) in some of the transgenic mice compared to the respective Wild Type (WT) mice. For the WT the average fertility rate ranged from 80% (C57BL/6), 90% (FVB/N), 45% (129Sv/J x C57Bl/6)F1 and 43% (CD1). For transgenic mice it was 52% (C57BL/6), 65% (FVB/N), 22% (129Sv/J x C57Bl/6)F1 and 25% (CD1).

Novel strategies targeting pathogen transmission reduction in insect vectors: Tsetse‐transmitted trypanosomiasis control

AbstractInsect vectors are essential for the transmission of important human diseases such as malaria, leishmaniasis, Chagas and sleeping sickness. Insects are also responsible for the transmission of agricultural diseases that affect livestock and crops. Traditionally, control of the vector populations has been an effective disease management strategy. Recently, vector control strategies have been fortified by research in insect biology and in insect–pathogen interactions as well as by the development of transgenic technologies. In addition to insect population reduction methods, disease control via selective elimination of pathogens in insects can now be explored. Here we explore the tsetse vectors of African trypanosomes and describe the application of recent knowledge gained in their symbiotic, reproductive and vectorial biology to develop novel disease control strategies.

Thirty years of plant transformation technology development

Technology development is seminal to many aspects of basic and applied plant transgenic science. Through the development and commercialization of genetically modified crops, the evolution of plant transgenic technologies is also relevant to society as a whole. In this study, literature statistics were used to uncover trends in the development of these technologies. Publication volume and impact (citation) over the past 30 years were analysed with respect to economic zones, countries, species and DNA delivery method. This revealed that, following a dramatic expansion in the 1980s, publications focusing on the development of transgenic technology have been slowing down worldwide since the early mid-1990s, except in a few leading Asian countries. The implications of these trends on the future of plant transgenic science as a whole are discussed.

Approaches to modeling stromal-epithelial interactions.

PURPOSE Techniques that can be used to examine the molecular mechanisms of stromal-epithelial interactions are described. MATERIALS AND METHODS A historical perspective of available techniques is provided. Recent developments and examples are used to illustrate and provide descriptive literature references for these methods. Since the possibilities for manipulating experimental systems are enormous and rapidly expanding, the reader should be aware that this review is an overview of how data have been and could be obtained rather than a comprehensive listing of what has been achieved. This review focuses on studies performed in the organs of the urogenital tract to illustrate techniques that are available.RESULTS Recent technological innovations have impacted our ability to manipulate specific components and pathways of stromal-epithelial interactions. They include rapid developments in transgenic and gene knockout mouse technology, and the development of highly efficacious gene delivery and expression systems. CONCLUSIONS These technologies have the potential to transform our understanding of the mechanistic basis of intercellular communication and point the way toward new therapeutic approaches for benign and malignant proliferative conditions.

Studying RNA editing in transgenic chloroplasts of higher plants.

RNA editing in plant cell organelles is a posttranscriptional process that changes the identity of individual nucleotides by pyrimidine transitions. C-to-U conversions are the predominantly found modifications in both mitochondrial and chloroplast transcripts of vascular plants. Research on functional, mechanistic, and evolutionary aspects of plant RNA editing has benefited immensely from the development of transgenic technologies for higher plant chloroplasts. This chapter describes methods for the generation of plants with stably transformed chloroplast genomes and their use for studying RNA editing processes in vivo.

Transgenic mouse models for studying the role of cartilage macromolecules in osteoarthritis.

The development of transgenic technology has made possible the generation of targeted gene-mutated mouse lines suitable for use in experimental osteoarthritis (OA) research. Transgenic mice harbouring mutations in cartilage collagen types II and IX develop early-onset OA and are therefore promising models of age-related OA, even though the mice often show signs of chondrodysplasia. Also, mouse lines harbouring other engineered mutations of the extracellular molecules have given rise to early OA. The molecular background of a few spontaneous mutations in mice has also been clarified and the characterization of the OA phenotype is now in progress. These mutations cause severe chondrodysplasia and death in homozygous mice, but the heterozygous offspring develop the early-onset OA phenotype.

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization.

Expression of Green Fluorescent Protein in Oligodendrocytes in a Time- and Level-Controllable Fashion with a Tetracycline-Regulated System

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.

Dynamic Role of Extracellular Matrix Metalloproteinases in Heart Failure

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is extensive myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs) which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation during heart failure. There is no known program which has declared its aim the investigation of regulation of fibrosis in hypertrophy and disruption of ECM in cardiac dilatation and failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or down regulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor β1 (TGFβ), decorin, collagen, and integrins in heart failure.

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-beta1 (TGF-beta), decorin, and collagen in cardiomyopathy and heart failure.

Gene targeting in embryonic stem cells: the new physiology and metabolism.

The development of transgenic technology, whereby genes (or mutations) can be stably introduced into the germline of experimental mammals, now allows investigators to create mice of virtually any genotype and to assess the consequences of these mutations in the context of a developing and intact mammal. In contrast to traditional "gain-of-function" mutations, typically created by microinjection of the gene of interest into the one-celled zygote, gene targeting via homologous recombination in pluripotential embryonic stem cells allows one to modify precisely the gene of interest. The purpose of this review is to introduce the reader to the history of development of embryonic stem cell technology, the current methods employed to create "knock-out" mice, and the application of these methods to solve problems in biology. While the technology promises to provide enormous insight into mammalian development genetics, our desire is that this review will stimulate the application of gene targeting in embryonic stem cells to begin to unravel problems in complex regulatory pathways, specifically intermediary metabolism and physiology.