Genetic Engineering Research Research Articles

Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin)

Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

Relación de los cultivos modificados genéticamente con el ambiente y la salud de la población costarricense

Genetic engineering and the food derived from genetically modified crops (GMCs) have been the center of debate worldwide, as has occurred historically with the advent of new technologies. Questions are derived from the potential impact of GMCs to the environment and the safety of the products to the consumers. In relation to the first inquiry, practice has been oriented to a case-by-case-study, according to the own characteristics of the GMC, in order to minimize its impact in the environment. Scientific studies in diverse latitudes of the world have demonstrated that GMCs in the market showed no adverse effects related to this issue. In relation to food derived from the GMCs, rigorous evaluation protocols have been developed and approved by FAO and WHO to guarantee the innocuousness of these products. Up to the moment, no contraindications for human health have been pointed out for the products that are available today in the market. In the particular case of Costa Rica, the country has established since the 90s a regulatory biosafety framework for the management of the GMCs, safeguarding the biodiversity of the country and the health of consumers. At the same time the country has made significant public and private investments in the field that allowed the country to obtain a leading position in biosafety in the region and genetic engineering research at national research centers. Any attempt to restrict or prohibit these activities in the country, will put in risk the previously described investment, will affect the generation of new knowledge for decision making and the leadership in the field, preventing the benefits derived from this promising technology.

Induction of Direct Adventitious Shoot Regeneration in Pear (Pyrus communis L.)

Efficient direct shoot regeneration of two pear (Pyrus communis L.), namely 'bartlett' and 'dargazi' was successfully developed for the use in future genetic engineering research. The basal MS and NN media supplemented with different concentrations of TDZ (0, 2.5, 5, 7.5 ?M) or BAP (0, 4, 8, 16 ?M) in combination with NAA (1 ?M) were compared. The result showed that direct adventitious shoot regeneration in pear was highly dependent on genotype, explant type and culture media. 'dargazi' had higher rate of regeneration compared to 'bartlett' and in both cultivars the highest per cent of regeneration was observed in lower sections of the leaves. Although the highest per cent of regeneration in 'bartlett' (38) was attained in the NN medium containing 2.5 µM TDZ and 1 µM NAA, but the differences in shoot regeneration between media containing 5 or 7.5 µM TDZ and 1 µM NAA were not significant. The highest per cent of regeneration in 'dargazi' (56) was obtained in NN medium containing 7.5 µm TDZ and 1 µm NAA. Plant Tissue Cult. & Biotech. 24(1): 87-92, 2014 (June) D. O. I. http://dx.doi.org/10.3329/ptcb.v24i1.19215

Immune modulation of the brain-gut-microbe axis.

1 Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland 2 Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Sadat City, Egypt 3 Department of Psychiatry, University College Cork, Cork, Ireland 4 Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland *Correspondence: s.elaidy@ucc.ie

Stem cells' guided gene therapy of cancer: New frontier in personalized and targeted therapy.

Diagnosis and therapy of cancer remain to be the greatest challenges for all physicians working in clinical oncology and molecular medicine. The statistics speak for themselves with the grim reports of 1,638,910 men and women diagnosed with cancer and nearly 577,190 patients passed away due to cancer in the USA in 2012. For practicing clinicians, who treat patients suffering from advanced cancers with contemporary systemic therapies, the main challenge is to attain therapeutic efficacy, while minimizing side effects. Unfortunately, all contemporary systemic therapies cause side effects. In treated patients, these side effects may range from nausea to damaged tissues. In cancer survivors, the iatrogenic outcomes of systemic therapies may include genomic mutations and their consequences. Therefore, there is an urgent need for personalized and targeted therapies. Recently, we reviewed the current status of suicide gene therapy for cancer. Herein, we discuss the novel strategy: genetically engineered stem cells' guided gene therapy. Stem cells have the unique potential for self renewal and differentiation. This potential is the primary reason for introducing them into medicine to regenerate injured or degenerated organs, as well as to rejuvenate aging tissues. Recent advances in genetic engineering and stem cell research have created the foundations for genetic engineering of stem cells as the vectors for delivery of therapeutic transgenes. Specifically in oncology, the stem cells are genetically engineered to deliver the cell suicide inducing genes selectively to the cancer cells only. Expression of the transgenes kills the cancer cells, while leaving healthy cells unaffected. Herein, we present various strategies to bioengineer suicide inducing genes and stem cell vectors. Moreover, we review results of the main preclinical studies and clinical trials. However, the main risk for therapeutic use of stem cells is their cancerous transformation. Therefore, we discuss various strategies to safeguard stem cell guided gene therapy against iatrogenic cancerogenesis. Defining cancer biomarkers to facilitate early diagnosis, elucidating cancer genomics and proteomics with modern tools of next generation sequencing, and analyzing patients' gene expression profiles provide essential data to elucidate molecular dynamics of cancer and to consider them for crafting pharmacogenomics-based personalized therapies. Streamlining of these data into genetic engineering of stem cells facilitates their use as the vectors delivering therapeutic genes into specific cancer cells. In this realm, stem cells guided gene therapy becomes a promising new frontier in personalized and targeted therapy of cancer.

Analysis of the activity of virus internal ribosome entry site in silkworm <italic>Bombyx mori</italic>

Internal ribosome entry site (IRES) has been widely used in genetic engineering; however, the application in silkworm (Bombyx mori) has hardly been reported. In this study, the biological activity of partial sequence of Encephalomyocarditis virus (EMCV) IRES, Rhopalosiphum padi virus (RhPV) IRES, and the hybrid of IRES of EMCV and RhPV were investigated in Spodoptera frugiperda (Sf9) cell line and silkworm tissues. The hybrid IRES of EMCV and RhPV showed more effective than EMCV IRES or RhPV IRES in promoting downstream gene expression in insect and silkworm. The activities of all IRESs in middle silk gland of silkworm were higher than those in the fat body and posterior silk gland. The hybrid IRES of EMCV and RhPV was integrated into silkworm genome by transgenic technology to test biological activity of IRES. Each of the positive transgenic individuals had significant expression of report gene EGFP. These results suggested that IRES has a potential to be used in the genetic engineering research of silkworm.

Isolating Barley (Hordeum vulgare L.) B1 Hordein Gene Promoter and Using Sequencing Analaysis For The Identification of Conserved Regulatory Elements By Bioinformatic Tools

Gene expression is a complex multi-step process. For the efficient expression of foreign genes in plants, it is essential to optimize every step of the process for the plant machinery, which includes choosing suitable promoters. Promoters play the most important role in determining the temporal and spatial expression pattern and transcript level of a gene. Some strong constitutive promoters, such as cauliflower mosaic virus 35s promoter, are widely used in plant genetic engineering research. However, the expression levels of the foreign genes in all tissues are often unsatisfied, but some of such problems can be may solved by using a strong seed-specific promoter to restrict gene expression to the seed only. The aim of this article was to characterize a B1 hordein-specific promoter. The promoter region of B1 hordein gene was isolated from the genomic DNA of Walfajre and Alger barley by polymerase chain reaction. Sequence analysis showed that the cloned fragment B hordein promoter (BHP) - contained motifs like TATA box, (CA)n box, ACGT motif, AAAG motif, GCN4-like motif and Ebox, which constituted the seed-specific promoter activity. It was therefore concluded that B1 hordein promoters can be used to engineer and subsequently study stable seed specific gene expression in barley, and potentially to modify barley seeds through genetic engineering. Keywords: Barley, Seed Specific Promoter, Motif, Hordein African Journal of Biotechnology Vol. 11(29), pp. 7378-7387, 10 April, 2012

Research and adoption of biotechnology strategies could improve California fruit and nut crops

California's fruit and nut tree crops represent one-third of the state's cash farm receipts and 70% of U.S. fruit and nut production. Advances in crop biotechnology and genetic engineering could help protect these valuable crops from pests and diseases and improve productivity. However, due to the difficulty of genetically engineering woody tree crops, as well as intellectual property concerns, regulatory hurdles and public perceptions about genetic engineering, biotechnology has not gained a foothold in this area of agriculture. Our survey of published genetic engineering research and issued field trial permits between 2000 and 2011 revealed that citrus and grape are the focus of most current work, and that walnut — not the more widely planted almond — is the focus among nut crops. Matching publicly funded genetic engineering research projects to a survey of the industry's top needs, we found that far less than half of the funded research has focused on the top-identified pest and disease threats. The most promising genetic engineering technology for fruit and nut tree crops may be transgrafting, which could address consumer concerns and benefit growers.

Genetic engineering and enzyme research in lignocellulosic ethanol production

Genetic engineering and enzyme research in lignocellulosic ethanol production

Research in tree genetic engineering; the Canadian context for field trial of GM trees

Canada is in a unique position for the regulation of GM (genetically modified) plants since it is based on the novelty of the resulting traits and not on the process to obtain it. For this reasons the term “plant with novel traits” (or PNTs) is used and these traits can be introduced into plants using biotechnology, mutagenesis or conventional breeding techniques. In Canada, less than a handful of GE tree field trials have been conducted. In all cases, poplars or spruces were used. Consistent with international guidance provided for living modified organisms, particular attention is given to a specific trait by considering both its novelty for the Canadian environment and its potential effect on human health and the environment. Our research is in line with the development of a consolidated research approach and knowledge needed for international guidance (e.g. Cartagena Protocol on Biosafety under the Convention on Biological Diversity) developed for genetically modified organisms. Important losses due to insects and fungal pathogens result in a considerable reduction of yield in Canadian forest productivity. Compounding these problems is the increased movement of pests that has been facilitated by increased international trade of agricultural and forest products as well as global climate change. More recently the increased need for biomass production and specific bioproducts to sustain crop-based biofuel production has emerged. Forest biotechnology has the potential to maintain forest sustainability by contributing to the transformation of our forest industry so as to improve productivity in the context of tree plantation. In this presentation, we will show how genetic engineering of forest trees has been used in improving tree adaptation and pest protection. We will also describe the work being done on environmental assessment of field-grown trees and results on long-term expression of engineered genes.

What Is Human in Humans? Responses from Biology, Anthropology, and Philosophy

Genomics has brought biology, medicine, agriculture, psychology, anthropology, and even philosophy to a new threshold. In this new context, the question about "what is human in humans" may end up being answered by geneticists, specialists of technoscience, and owners of biotech companies. The author defends, in this article, the idea that humanity is at risk in our age of genetic engineering, biotechnologies, and market-geared genetic research; he also argues that the values at the very core of our postgenomic era bring to its peak the science-based ideology that has developed since the time of Galileo, Newton, Descartes, and Harvey; finally, it shows that the bioindustry has invented a new genomythology that goes against the scientific evidence produced by the research in human sciences in which life is interpreted as a language.

Sugarcane genetic engineering research in South Africa: From gene discovery to transgene expression

Over the past 15 years, recombinant DNA and in vitro culture technologies have been used in concert at the South African Sugarcane Research Institute (SASRI) to genetically engineer sugarcane. The purpose of such research has been two-fold, viz. to establish proof-of-principle regarding the delivery of novel input and resistance traits to sugarcane and to investigate the genetic basis of sucrose accumulation. Underpinning these goals has been the in-house development of essential genetic resources, including the isolation of appropriate transgenes and gene promoter elements, and the optimisation of transformation and tissue culture technologies. With regard to the latter, emphasis has been placed on the development of tissue culture strategies that minimise the potential for somaclonal variation, while maximising the efficiency of germplasm transformation, selection, regeneration and acclimation. The isolation of promoters to drive high-level and targeted transgene expression, as well as the identification of DNA sequences with the potential to deliver the desired phenotype, have been strongly driven by advances in gene discovery and expression technologies. Early approaches relied on the analysis of Expressed Sequence Tag libraries constructed from sugarcane tissues at different developmental stages or challenged with selected abiotic and biotic stress agents. Subsequent improvements in DNA subtractive hybridisation technologies (e.g. suppression subtractive hybridisation) and gene expression analysis (e.g. Affymetrix® Sugarcane Genome GeneChip) provided new and powerful means for gene and promoter discovery. Reviewed here are the advances in in vitro culture, gene discovery and transgene expression research, which have formed the foundation of the genetic engineering program at SASRI. Also discussed are the opportunities and challenges in these areas of research that are presented by the availability of ultra-high throughput DNA sequencing (e.g. the 454 Sequencing System) for transcriptome analysis and technologies for rapid and accurate profiling of the sugarcane metabolomes.

The Pandora’s Box

The medical problem, more technical in nature, of modifying the defective gene(s) in an individual gamete or zygote by gene therapy and implanting the replaced or repaired genes into the mother thereby producing a healthy child have not yet been surmounted. However, assuming such protocols can be successfully performed, gene therapy will probably be sanctioned universally by health authorities as a legitimate implementation of the mandate on physicians to treat the disabled. Another argument favoring the permissibility of gene therapy or genetic manipulation is the fact that the gamete or even the fertilized zygote is not a person per se. Thus, gene manipulation is not considered as tampering with an existing or even potential human being (foetus). One can also argue that any surgery performed on a live human being must certainly be permitted on a gamete or fertilized zygote. For example, if a cure for hemophilia, Tay Sachs disease or Huntington’s disease were possible, it should surely be permissible and promoted. It seems clear that genetic engineering and gene therapy can and should be used to treat, cure or even prevent diseases. But should these techniques be allowed to alter human traits such as eye color, height, personality, intelligence and facial features? Obviously not. Should we create piano players with three hands? Should we create supermicroorganisms for agricultural purposes? Perhaps, but they may also be used for germ warfare and should therefore be discouraged. To attempt to clone a human being is not legally prohibited in many cultures (Judaism) but is probably morally inappropriate. The possible deleterious effects of genetic engineering and gene therapy are not yet fully known. Can such genetic manipulation unmask inactive cancer gene or alternatively, activate tumor suppressor genes? In addition to the medical and scientific aspects of genetic engineering and DNA recombinant research, the spiritual and theological aspects also require justification. Genetic engineering in medicine has been long researched for transplantation purposes, for example, to make organs or body parts like valves for the heart from pigs. There are still safety concerns about large organ transfer from other species (xenotransplants). The most controversial form of genetic engineering in medicine is the use of cloning technology to create organs for transplantation purposes so that they are immunologically compatible. There are fears that it could be misused for cloning human beings or making genetically enhanced “designer babies”, so that parents can select, chose and improve the characteristics of their babies like blue eyes, fair skin, tallness, gender etc. However, the success rate of cloning is very low and its applications are still in very early stages of research. These concerns are based on the doubts about the technology, its potentiality, newness, and applicability to all life forms. The people in favor of this technology think that genetic modification provides a great opportunity for solving hunger, food insecurity and malnutrition in the world since it can be adapted to all environmental conditions and help in increasing quantity and quality of food. At the same time, there are fears raised about the safety of the food in eating and also risks to ones health since it is considered as a new technology and people fear that some “rogue” genes will be transmitted to them. Also many people think genetic engineering is unnatural and call it as Franken foods and genetic modification will result in “superweeds” resistant to herbicides. The intrinsic concerns on this technology at large are based on how people view life, nature, religion, their personal emotions and values. There is a feeling that jumbling with genes in the organisms for our use is “playing God” and human beings should not intervene in God’s realm. Crossing natural species boundaries is creation of new life forms and inventing a new world through technology. However, at the same time we can say that high tech medicines involves playing with God and agriculture was started by disrupting nature. Also hybrid plants and animals like mules are cross-species organisms and they do exist in nature. In fact, mules have been cloned and can reproduce in that way!

Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

BackgroundWhite-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.ResultsThis paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.ConclusionThese findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.

Review of quantitative and qualitative studies on U.S. public perceptions of synthetic biology

How are public perceptions towards synthetic biology likely to evolve? Which factors will impact the framing of this emerging technology, its benefits and risks? The objective of this article is not to draw exhaustive conclusions about public perceptions of synthetic biology, but rather to provide readers with a review of integrated findings from the first quantitative and qualitative research ever conducted on this subject in the United States. Synthetic biology survey research shows two clear findings. The first is that most people know little or nothing about synthetic biology. Second, notwithstanding this lack of knowledge, respondents are likely to venture some remark about what they think synthetic biology is and the tradeoff between potential benefits and potential risks. Finding only some support for the “familiarity argument”—according to which support for emerging technologies will likely increase as awareness of them develops—this article suggests that analogs to cloning, genetic engineering and stem cell research appear to be recurrent in the framing process of synthetic biology. The domain of application seems to be another decisive factor in the framing of synthetic biology. Finally, acceptance of the risk-benefit tradeoff of synthetic biology seems to depend on having an oversight structure that would prove able to manage unknowns, human and environmental concerns, and long-term effects. The most important conclusion of this study is the need for additional investigation of factors that will shape public perceptions about synthetic biology, its potential benefits, and its potential risks.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-009-9035-6) contains supplementary material, which is available to authorized users.

Scientific linkage of science research and technology development: a case of genetic engineering research

In this study, the author tried to demonstrate the linkage between science research and technology development through non-patent citation analysis to reveal that the important knowledge resources from science research had significant impact on technology development. Genetic engineering technology was the field examined in this study. From the references listed in the patents, it was observed that the technology development in genetic engineering was influenced heavily by the research done by public sector. Over 90% of the citations were non-patent literatures, and the majority of non-patent citations were journal articles. Citing preferences, such as country preference and institute preference were observed from the data included in this study.

Gateway vectors for plant transformation

The availability of complete plant genome sequences has opened a new era of plant molecular research using approaches such as comprehensive expression analysis, ectopic expression and gene knockdown. For such purposes, new tools realizing easy and rapid construction of recombinant plasmids are required. Here, we describe Gateway cloning compatible binary vectors for post-genomic research in plant genetic engineering. The vectors comprise a variety of reporters, epitope tags and selective markers that should make them useful for construction of plasmids for Agrobacterium- mediated transformation of plants. We also describe vectors for promoter swapping using Gateway cloning technology.

Transgenic crops: Current challenges and future perspectives

The history of transgenic crops at present can be divided in two parts. The first era deals with the development of Genetically Modified (GM) crops. As the time went on, various social, political, environmental and technical issues related to transgenic crops took their birth. The development of transgenic crops has raised some issues more especially the problem of food and environmental safety, some technical impacts like effect on non target organisms, development of cross pest resistance, use of selectable marker genes, etc. There exists a thought that the pace of research in genetic engineering of crop plants may some day lead to the development of variations that will not ensure the survival of living creatures including human beings. Most of such concerns are just psychological and are often based on fear of negative political fall out or media coverage. The genetic engineering of crop plants is now moving towards the course of correction. It is the responsibility of concerned researchers to interpret such hazards and their solutions on technical basis and, therefore, establish a based line of acceptance for transgenic crops to the consumers.

Improving the Content of Essential Amino Acids in Crop Plants: Goals and Opportunities

The inability of humans and many farm animals to synthesize certain amino acids has long triggered tremendous interest in increasing the levels of these so-called essential amino acids in crop plants. Knowledge obtained from basic genetic and genetic engineering research has also been successfully