Transgenic Research Research Articles

Genome-Wide Identification, Characterization, and Stress-Responsive Expression Profiling of Genes Encoding LEA (Late Embryogenesis Abundant) Proteins in Moso Bamboo (Phyllostachys edulis).

Late embryogenesis abundant (LEA) proteins have been identified in a wide range of organisms and are believed to play a role in the adaptation of plants to stress conditions. In this study, we performed genome-wide identification of LEA proteins and their coding genes in Moso bamboo (Phyllostachys edulis) of Poaceae. A total of 23 genes encoding LEA proteins (PeLEAs) were found in P. edulis that could be classified to six groups based on Pfam protein family and homologous analysis. Further in silico analyses of the structures, gene amount, and biochemical characteristics were conducted and compared with those of O. sativa (OsLEAs), B. distachyon (BdLEAs), Z. mays (ZmLEAs), S. bicolor (SbLEAs), Arabidopsis, and Populus trichocarpa. The less number of PeLEAs was found. Evolutionary analysis revealed orthologous relationship and colinearity between P. edulis, O. sativa, B. distachyon, Z. mays, and S. bicolor. Analyses of the non-synonymous (Ka) and synonymous (Ks)substitution rates and their ratios indicated that the duplication of PeLEAs may have occurred around 18.8 million years ago (MYA), and divergence time of LEA family among the P. edulis-O. sativa and P. edulis–B. distachyon, P. edulis-S. bicolor, and P. edulis-Z. mays was approximately 30 MYA, 36 MYA, 48 MYA, and 53 MYA, respectively. Almost all PeLEAs contain ABA- and (or) stress-responsive regulatory elements. Further RNA-seq analysis revealed approximately 78% of PeLEAs could be up-regulated by dehydration and cold stresses. The present study makes insights into the LEA family in P. edulis and provides inventory of stress-responsive genes for further functional validation and transgenic research aiming to plant genetic improvement of abiotic stress tolerance.

Transient expression of the enhanced green fluorescence protein (egfp) gene in Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta) with three exogenous promoters

:A stable transformation system for Tetraselmis subcordiformis had been established, but a low transformation rate limits its application to this alga. As a promoter is a key factor for transgenic research, a suitable promoter could increase the transformation rate. To improve a transformation system for T. subcordiformis, three commonly used promoters for algal transformation were selected. The CMV, CaMV35S and SV40 promoters were transferred to T. subcordiformis by glass-bead agitation and particle bombardment, with the enhanced green fluorescence protein (egfp) gene as the reporter gene. The transformation rate was calculated according to the GFP fluorescence in transformed algae. Results showed that the CaMV 35S promoter transformation efficiency was higher than that of CMV and SV40 promoters. This report will contribute to efforts to establish a high-efficiency transformation system and expands the fundamental research and applications of this alga.

Purification and characterization of a novel heterodimer protease inhibitor from Streptomyces spp. VL J2 with potential biopesticidal activity against H. armigera

A protease inhibitor (PI) was isolated and purified from halo-alkaliphilic Streptomyces spp. VL J2. SDS-PAGE of purified PI revealed it to be a heterodimer of two unidentical subunits of 27.5 and 11.08kDa, which corroborates well with intact molecular mass of 38.5kDa obtained by GPC and MALDI −TOF. Inhibitory activity was confirmed by activity staining and reverse zymogram studies and the inhibitor was found to retain activity at <50°C and pH 2–9.5. It showed presence of two isoforms with isoelectric point of 5.5 and 5.7. The inhibition of trypsin and chymotrypsin indicated it to be a serine protease type belonging to serpin family. The stoichiometry of trypsin-inhibitor interaction was 1:2. Modification of amino acid showed presence of arginine and free sulfhydryl group at active site. Kinetic studies revealed the non-competitive type of inhibition of trypsin with low Ki value (9.4×10−9M). Activity against H. armigera showed significant decline and delay in larval (51%), pupal weights and periods, respectively, prominent physical abnormalities and reduced nutritional indices as a function of treatment. The results suggest that the purified PI has promising pesticidal activity and could serve as a potential candidate gene for transgenic plant research.

Assessing zygosity in progeny of transgenic plants: current methods and perspectives.

Homozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T1 generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labor intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, digital PCR and loop-mediated isothermal amplification also provide a cost effective, fast and not so labor intensive substitute of current methods. In this review, we have attempted to compare and contrast all the available efficient methods to distinguish homozygous plants in progeny of transgenics. This review also provides information of various techniques available for determining zygosity in plants so as to permit researchers to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plants from the laboratory to the field.

Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression.

The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5′ RACE and 3′ RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits.

Malatesta's Transgenic Research: Citation Analysis and Relevant Evidence

Malatesta's Transgenic Research: Citation Analysis and Relevant Evidence

Assessment of new scientific elements supporting the prolongation of prohibition of the placing on the market of genetically modified oilseed rape GT73 for food and feed purposes in Austria

Assessment of new scientific elements supporting the prolongation of prohibition of the placing on the market of genetically modified oilseed rape GT73 for food and feed purposes in Austria

Assessment of new scientific elements supporting the prolongation of prohibition of the placing on the market of genetically modified oilseed rape Ms8, Rf3 and Ms8×Rf3 for food and feed purposes in Austria

Assessment of new scientific elements supporting the prolongation of prohibition of the placing on the market of genetically modified oilseed rape Ms8, Rf3 and Ms8×Rf3 for food and feed purposes in Austria

Western blot detection of PMI protein in transgenic rice

Phosphomannose isomerase (PMI) encoding gene manA is a desirable selective marker in transgenic research. Understanding of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain (about 0.08 mg). PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Transgenic NfFeSOD Sedum alfredii plants exhibited profound growth impairments and better relative tolerance to long-term abiotic stresses

Transgenic research was preformed by transferring a cyanobacterial (Nostoc flagelliforme) iron superoxide dismutase gene (NfFeSOD) into heavy metal hyperaccumulator Sedum alfredii via Agrobacterium-mediated method. Beyond expectation, NfFeSOD-overexpressing S. alfredii plants exhibited profound impairments, including plant growth retardation, abnormal root architecture, and reduced leaf greenness, photosynthetic efficiency and metal accumulation efficiency. Although transgenic plants appeared physiologically sensitive to high temperature, a higher relative biomass growth was still observed under long-term high temperature and osmotic stresses. Further investigation found that reactive oxygen species (ROS) homeostasis of transgenic plants was significantly affected, being ~50 % reduction of H2O2 level relative to wild-type plants. Gene transcription including ROS responsive genes was overall attenuated in transgenic plants, being more significant at normal temperature than at high temperature. In addition, ascorbate peroxidase (APX) activity was increased nearly twofolds in transgenic plants as compared to wild-type control. It may be inferred that ectopic NfFeSOD overexpression gives rise to a substantial increase of APX activity and leads to a sharp reduction of H2O2 level, thus impairing basal ROS signaling and plant growth. Specific genetic background of S. alfredii may be responsible for this sharp reduction of H2O2 level induced by NfFeSOD overexpression. S. alfredii plant has acclimated to elevated levels of ROS induced by heavy metals in native habitats and should require high ROS levels for basal signaling. We thus suppose that a sustained disturbance of high basal ROS signaling in metal hyperaccumulators may instead incur very sensitive response and thus result in profound growth impairments.

Genetic engineering for peanut improvement: current status and prospects

Peanut is an important source of edible oil, dietary minerals, and vitamins, and proteins. Environmental stresses like biotic and abiotic factors are main constraints for peanut production and productivity. Generation of resistant varieties against these stresses can lead to quantum leap in the crop productivity. Potential biotechnology approaches through engineering and expression of novel genes can improve stress tolerance/resistance mechanisms that ultimately lead to crop adaptation and yield enhancement. There are several genetic engineering techniques are adopted, among them Agrobacterium mediated and biolistic methods are widely employed for transforming peanut varieties. Expression of abiotic stress related genes or transcription factors like DREB, PDH45, NAC, mtlD, NHX etc. in different peanut varieties resulted in enhanced tolerance to drought, salinity, temperature extremes and osmotic adjustments. In addition, expression of transgene could regulate the biochemical pathways which lead to scavenge free radicals, lipid peroxidation, increase photosynthetic efficiency, water use efficiency and transpiration rate. To address the biotic stresses, genes like glucanase, chitinase, chloroperoxidase, coat proteins, crystal proteins, and pathogen related genes have been expressed in peanut which exhibited enhanced resistance towards different fungal, bacterial pathogens and pests. Interestingly, plant based edible vaccines and therapeutic antibodies are produced in plants through the expression of urease B and oleosin to combat chronic infections. Allergy is a prime concern in peanut that is to be inhibited through RNA interference mechanisms. We review here factors influencing the transformation, recent progress in peanut transgenic research, development and future perspectives.

Genetically engineered livestock for agriculture: a generation after the first transgenic animal research conference.

At the time of the first Transgenic Animal Research Conference, the lack of knowledge about promoter, enhancer and coding regions of genes of interest greatly hampered our efforts to create transgenes that would express appropriately in livestock. Additionally, we were limited to gene insertion by pronuclear microinjection. As predicted then, widespread genome sequencing efforts and technological advancements have profoundly altered what we can do. There have been many developments in technology to create transgenic animals since we first met at Granlibakken in 1997, including the advent of somatic cell nuclear transfer-based cloning and gene editing. We can now create new transgenes that will express when and where we want and can target precisely in the genome where we want to make a change or insert a transgene. With the large number of sequenced genomes, we have unprecedented access to sequence information including, control regions, coding regions, and known allelic variants. These technological developments have ushered in new and renewed enthusiasm for the production of transgenic animals among scientists and animal agriculturalists around the world, both for the production of more relevant biomedical research models as well as for agricultural applications. However, even though great advancements have been made in our ability to control gene expression and target genetic changes in our animals, there still are no genetically engineered animal products on the market for food. World-wide there has been a failure of the regulatory processes to effectively move forward. Estimates suggest the world will need to increase our current food production 70% by 2050; that is we will have to produce the total amount of food each year that has been consumed by mankind over the past 500years. The combination of transgenic animal technology and gene editing will become increasingly more important tools to help feed the world. However, to date the practical benefits of these technologies have not yet reached consumers in any country and in the absence of predictable, science-based regulatory programs it is unlikely that the benefits will be realized in the short to medium term.

Genetically engineered livestock for biomedical models.

To commemorate Transgenic Animal Research Conference X, this review summarizes the recent progress in developing genetically engineered livestock species as biomedical models. The first of these conferences was held in 1997, which turned out to be a watershed year for the field, with two significant events occurring. One was the publication of the first transgenic livestock animal disease model, a pig with retinitis pigmentosa. Before that, the use of livestock species in biomedical research had been limited to wild-type animals or disease models that had been induced or were naturally occurring. The second event was the report of Dolly, a cloned sheep produced by somatic cell nuclear transfer. Cloning subsequently became an essential part of the process for most of the models developed in the last 18years and is stilled used prominently today. This review is intended to highlight the biomedical modeling achievements that followed those key events, many of which were first reported at one of the previous nine Transgenic Animal Research Conferences. Also discussed are the practical challenges of utilizing livestock disease models now that the technical hurdles of model development have been largely overcome.

Advances in genetic engineering of the avian genome: "Realising the promise".

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac + Cry2A, GT gene and insect mortality

Expression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production.

Transgenic Research in Fruit Crops: Current Status

Gene mining for various biotic and abiotic stresses could be utilized only when we can transfer them to our important crops so that a large number of the population can be benefitted by this. In this light gene transfer through various methods can be done and it will be more useful where traditional breeding approach is not feasible. However, transgenic research in fruit crops is very low so that present information will be utilized by fruit breeders to a design future program for developing transgenic that ultimately open the way for those crops where gene transfer is difficult through conventional breeding.

Promoter analysis of bovine cardiomyopathy-associated protein 1 gene.

The CMYA1 (cardiomyopathy-associated protein 1) is an actin-binding protein that plays a vital role in cardiac morphogenesis. CMYA1 is expressed specifically in the myocardial and skeletal muscle and is up-regulated in injured muscle. We therefore speculated that the bovine CMYA1 promoter might be muscle-specific. In this study, the promoter (+20/-1135) region of the bovine CMYA1 gene was cloned into a pEGFP-1 vector, and we found that the EGFP was observed only in C2C12 and myoblast cells. Thus, the CMYA1 promoter is muscle-specific. Thereafter, eight pGL3-basic vectors with various truncated CMYA1 promoter fragments were transfected into C2C12 cells, to identify the core promoter region using a Dual-Luciferase Reporter Assay System. The results showed that the promoter region from -457 to +20 bp was essential for CMYA1 to maintain the promoter activity, implying that this region may be the CMYA1 core promoter. We thus illustrate that the core promoter is muscle-specific. To evaluate the activity of the CMYA1 core promoter, the CMYA1 core and muscle creatine kinase (MCK) promoters were cloned into a pcDNA3.1 vector. The expression levels of their target genes were measured in C2C12 cells using real-time polymerase chain reaction. The CMYA1 promoter drove the expression of the target gene six times higher than did the MCK promoter. The results thus suggest that the CMYA1 promoter could be an effective muscle-specific promoter, which may be useful in further studies of cardiomyopathy treatment and transgenic animal research.

Veteriner Tedavilerinde Kök Hücre Araştırmaları ve Uygulamaları

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Times New Roman'; min-height: 15.0px} p.p2 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px 'Times New Roman'} span.s1 {font: 12.0px 'Times New Roman'} Stem cells, a promising hope for regenerative medicine with a keen scientific focus and getting an immense priority in research as using in cloning, gene targeting, drug testing and deliverance, transgenic rese-arch, and cellular therapy to patient. Having potentiality for self-renewal and differentiation into various kinds of body cells with diversified functions, these naive cells of the body are scholastically using for treating human and animals in various chronic, notorious and devastating diseases. With the advancement of science and tech-niques, stem cells become an outstanding tool for regenerative therapy in veterinary medicine. Due to having some notable beneficial characters of Mesenchymal stem cells over embryonic one, like; ease of harvesting and application, lower immunogenic responses, no evidence of teratoma formation showing higher preferences for their uses. There are proofs of appliance of stem cells for tendinitis, cartilage injury, osteoarthritis, spinal injury, skin healing, cardiac problem, bone fracture, eye treatment, renal cure, and muscular therapy in animals. The visionary use of multipotent stem cells has shaped a new horizon for clinicians to deal with challenges and will bring a better hope for mankind. This review has spotlighted on the restorative application and victorious utiliza-tion of stem cells in animals.

Transgenic studies for modulating terpenoid indole alkaloids pathway in Catharanthus roseus: present status and future options

The anti-neoplastic herb Catharanthus roseus (Madagascar periwinkle; Family Apocynaceae), known to harbor >130 bioactive terpenoid indole alkaloids (TIAs), is in global attention of transgenic research for pathway engineering. This medicinal herb has a long folklore history of its usage in the treatment of several health disorders owing to the presence of a variety of secondary metabolites, particularly the alkaloids, phenolics and anthocyanins. Among its various bioactive molecules, C. roseus is most valued for being the exclusive bio-resource of two of the most effective anti-cancerous drugs—vincristine and vinblastine that are indispensible components of several modern day chemotherapeutic regimens. The in-planta yields of these bioactive TIAs are extremely low (<0.0002 %) owing to very rigid developmental, genetical and environmental regulation controls as a part of plant’s own auto-protection strategies against these cytotoxic molecules. Consequently the high cost of production coupled with increasing market demand for these alkaloidal drugs have made TIAs pathway as one of the preferred target for plant metabolic engineering efforts to boost their commercial production. The transgenic approaches so far applied in C. roseus can be grouped into three major lines of investigations. The first line of efforts have been dominated by the genetic transformation studies carried out with wild type Ti or Ri plasmids to measure the influence of their random insertion on TIAs biogenesis. These studies, besides helping the optimization of basic transformation protocols at different levels of cellular differentiation, have largely contributed towards the better resolution and expression of TIAs pathway architecture at the level of associated genes and enzymes. Second line of metabolic engineering works is being centered around the efforts on preparation of genomic libraries and genome mapping to hunt for TIAs pathway genes and associated transcription regulators/factors, followed by their cloning, insertion and hyper-expression to modulate the carbon flux towards desired end products. Modern day transgenic attempts in C. roseus are largely focused on superimposing the concurrent influences of biotic and abiotic elicitation, T-DNA activation and RNAi or virus induced gene silencing approaches for higher TIAs biogenesis. The pace of transgenic research in C. roseus in last two decade has been rapid and demands periodic compilation and reviewing of published literature to provide a handy status up-date of the subject. The present compilation is an effort in this direction and intends to provide a state-of-art account of various transgenic approaches currently being pursued in this high value herb. More than 130 research papers have appeared on various aspects of transgenic cells, tissues and plant production in C. roseus in 30 years. The bulk of these efforts (64.96 %) were made at the level of Agrobacterium rhizogenes-mediated hairy root cultures, followed by 26.80 % in A. tumefaciens-mediated transformed tissues and 8.24 % in cells/tissue cultures subjected to particle bombardment approach. In the present compilation, these studies are further grouped into specific categories wherein over-expression of TIAs pathway specific genes, transcriptional factor regulation, elicitation/stress superimposition, transgenic plant regeneration, metabolites profiling and gene silencing etc. were investigated in different transformed tissue.

Relevance of a new scientific publication (Trtikova et al., 2015) on previous EFSA GMO Panel conclusions on the risk assessment of maize MON 810 and other Cry1Ab‐expressing Bt‐maize events

Relevance of a new scientific publication (Trtikova et al., 2015) on previous EFSA GMO Panel conclusions on the risk assessment of maize MON 810 and other Cry1Ab‐expressing Bt‐maize events