Transgenic Protein Research Articles

Degradation of transgenic Bacillus thuringiensis proteins in corn tissue in response to post-harvest management practices.

Knowledge of the persistence of Cry proteins in transgenic corn residue after harvest is necessary to assess the ecological risk to nontarget organisms. The amount of protein remaining in crop residue declines over time by a combination of microbial decomposition and leaching, both influenced by temperature, precipitation, and the amount of residue-soil contact. Here, we investigated how long biologically active Cry proteins persist in SmartStax corn residue expressing Cry1A.105, Cry1F, Cry2Ab2, Cry3Bb1, and Cry34/35Ab1, when subjected to four post-harvest practices (chisel plow tillage, flail mowing, cover crop planting, and undisturbed residue). Protein activity in residue samples collected up to 25weeks after harvest was measured by Ostrinia nubilalis feeding bioassays and cross validated with detection frequencies determined by ELISA. All corn residue remained above ground in the flail-mowed and undisturbed treatments, while the cover crop and chisel plow treatments left 88.3 and 39.6% of the residue remaining above ground, respectively. Cry proteins retained biological activity for as long as 24weeks after harvest when residue was left above ground with less soil contact, typical of no-till corn systems. ELISA detections were positively correlated with results of the feeding bioassays, which revealed the presence of active proteins beyond the point of ELISA detection.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods

The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

Abstract 1919: Prediction of systemic cytokine exposure in human after IV administration of oncolytic myxoma virus, using quantitative systems pharmacology modeling

Abstract A quantitative systems pharmacology (QSP) model of oncolytic immunotherapies based on the myxoma virus (MYXV) platform was constructed to project systemic cytokine exposure after intravenous (IV) administration, and to determine safe doses. Oncolytic viruses selectively replicate in and lyse tumor cells and provide stimulation to the immune system. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins. The QSP model mechanistically describes virus administration, infection of competent cells, promoter-dependent gene transcription, cytokine payload production and secretion, and secondary cytokine responses. An in vitro model was calibrated to cytokine release data from virus infected PBMCs. A mouse model was developed to recapitulate virus PK data and cytokine data collected in the serum of tumor-bearing mice who received various IV doses of oncolytic virus constructs. The cytokine data include time-resolved measurements of transgenic cytokine expression (the viral payload), the immediate endogenous cytokine response to the virus vehicle itself, as well as secondary cytokine responses. Learnings from the in vitro model, the in vivo mouse model as well as from human specific literature information were used to create the human model. The resulting human model was used to project systemic expression of cytokine payloads and subsequent secondary cytokine responses for various IV doses of the oncolytic virus construct. Projected cytokine levels were compared to previously established cytokine exposure at maximum tolerated doses of IV administered cytokines to assess potential safety implications. The impact of uncertainties in model predictions stemming from variability in the available data and from the assumptions made during model building was evaluated. The upper range of predicted systemic cytokine exposure in humans is still expected to fall within known safety margins at the planned doses of oncolytic immunotherapies. In the absence of human data, QSP modeling represents a holistic approach of integrating all available knowledge and preclinical data for the purpose of human prediction. It yields valuable insight on the system-wide effects of IV oncolytic viral administration and can be expanded in the future to include assessment of efficacy in the form of tumor growth inhibition. Citation Format: A. Katharina Wilkins, Lina S. Franco, Diana H. Marcantonio, Karyn L. Sutton, Joshua F. Apgar, John M. Burke, Grant McFadden, Fei Hua, Leslie L. Sharp. Prediction of systemic cytokine exposure in human after IV administration of oncolytic myxoma virus, using quantitative systems pharmacology modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1919.

Abstract 1920: Armed oncolytic myxoma virus demonstrates transgene production, function, and therapeutic activity xenograft models

Abstract Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system, representing a promising therapeutic option in development to treat cancers that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Pox family of double stranded DNA viruses. The natural host of MYXV is a subset of rabbits and hares, but MYXV is able to infect cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins, making it an excellent oncolytic virus for introduction of immunomodulatory proteins. Viral reproduction rates, transgene production, transgene function, and inhibition of cell growth of multi-armed myxoma virus was evaluated in vitro using cell lines representing multiple human cancer indications. Efficacy and mechanism of action of multi-armed myxoma virus was also demonstrated in vivo via xenograft models. Multi-armed myxoma represents a novel, engineerable, oncolytic virus with preclinical characteristics that warrant further investigation as an immunomodulatory cancer therapeutic. Citation Format: Lina S. Franco, Mario Abrantes, Wazir Abdullahi, Ana L. de Matos, Benjamin S. Walker, Zachary Tacner, Cassandra Kien, Nicole Grigaitis, Grant McFadden, Steven J. Potts, Leslie L. Sharp. Armed oncolytic myxoma virus demonstrates transgene production, function, and therapeutic activity xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1920.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway.

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.

Effect of GM maize on metabolism and performance of chicken

Effect of GM maize on metabolism and performance of chicken

Chimeric antigen receptors (CARs): universal tools in adoptive cell therapy

The observation that tumor-infiltrating lymphocytes (TIL) after ex vivo amplification can control tumors in the long term led to the concept of redirecting patients' cytolytic T‑cells by areceptor with defined specificity against the tumor. Development of arecombinant receptor-signal molecule (chimeric antigen receptor, CAR) to increase selectivity and enhance anti-tumor immunity. Description of aprototype CAR, overview of the modular composition and further development of CAR technology for use in adoptive immune cell therapy. Intensive research over the last two decades has shown how CAR-mediated T‑cell activation is influenced by factors such as binding affinity, the epitope of the target antigen, its expression density and accessibility on the tumor cells, as well as by the signaling domains and their combination to induce T‑cell activation. The quality and duration of the T‑cell response can be specifically modulated by modifying the modular composition of the CAR; CAR T‑cells can act as "biopharmaceutical factories" (T-cells redirected for unrestricted cytokine-mediated killing, TRUCK) in the tissue by CAR-mediated release of transgenic therapeutic proteins. Adoptive CAR T‑cell therapy has shown clinical efficacy in the treatment of hematological malignancies; the treatment of solid tumors, however, is more challenging. Allogeneic CAR T‑cell technology is aimed at generating "off-the-shelf" CAR T‑cells that are accessible for alarge number of patients. Afurther promising approach is the use of CAR T‑cells for other therapeutic applications such as the treatment of autoimmune diseases.

Long-Term Follow-Up of Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy.

In 2009, cerebral adrenoleukodystrophy (c-ALD) became the first brain disease to be treated with lentiviral (LV)-based hematopoietic stem cell gene therapy with the ABCD1 gene in four boys (P1-P4) who had demyelinating lesions expected to be lethal in the short term and no bone marrow donor. We report the clinical and magnetic resonance imaging (MRI) follow-up over a mean of 8.8 years posttransplant. In parallel, vector genome copies, expression of transgenic ALD protein (ALDP), and viral integration sites were determined in peripheral blood cells. Prior to transplant, the four patients had a normal or near normal neurocognitive status but gadolinium-enhanced demyelination in various brain regions. Gadolinium diffusion disappeared during the first year posttransplant. P3 kept a near normal status until 8.3 years of follow-up, but P1, P2, and P4 showed major cognitive degradation around 9, 28, and 60 months posttransplant. Neurological status and demyelination stabilized until last evaluation in P2, but deteriorated in both P1 at 10 years and P4 at 3 years posttransplant. The proportion of myeloid and lymphoid cells expressing transgenic ALDP decreased by half within 5 years then stabilized around 5% to 10%. Integration site analysis revealed a durable polyclonal distribution of genetically corrected hematopoietic cells. No adverse effects were observed. The long-term arrest of demyelination at MRI and persistence of transduced hematopoietic progenitors support that LV gene therapy may be a safe and durable treatment of c-ALD. However, the neurological degradation observed in three out of four patients mitigates the benefit of this therapy, calling for an earlier intervention, more potent vectors, and additional therapeutic strategies.

Rat perichondrium transplanted to articular cartilage defects forms articular-like, hyaline cartilage

Abstract Objective Perichondrium autotransplants have been used to reconstruct articular surfaces destroyed by infection or trauma. However, the role of the transplanted perichondrium in the healing of resurfaced joints has not been investigated. Design Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112 days after surgery. Results Tracing of transplanted cells showed that both perichondrium and periosteum transplant-derived cells made up the large majority of the cells in the regenerated joint surfaces. Perichondrium transplants contained SOX9 positive cells and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive and Col1a1-negative chondrocytes and a matrix rich in proteoglycans. At later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. Periosteum transplants initially lacked SOX9 expression and despite a transient increase in SOX9 expression and chondrogenic differentiation, remained Col1a1 positive, and were continuously thinning as periosteum-derived cells were incorporated into the subchondral compartment. Conclusions Perichondrium and periosteum transplanted to articular cartilage defects did not just stimulate regeneration but were themselves transformed into cartilaginous articular surfaces. Perichondrium transplants developed into an articular-like, hyaline cartilage, whereas periosteum transplants appeared to produce a less resilient fibro-cartilage.

Immunomodulation in Administration of rAAV: Preclinical and Clinical Adjuvant Pharmacotherapies.

Recombinant adeno-associated virus (rAAV) has attracted a significant research focus for delivering genetic therapies to target cells. This non-enveloped virus has been trialed in many clinical-stage therapeutic strategies but important obstacle in clinical translation is the activation of both innate and adaptive immune response to the protein capsid, vector genome and transgene product. In addition, the normal population has pre-existing neutralizing antibodies against wild-type AAV, and cross-reactivity is observed between different rAAV serotypes. While extent of response can be influenced by dosing, administration route and target organ(s), these pose concerns over reduction or complete loss of efficacy, options for re-administration, and other unwanted immunological sequalae such as local tissue damage. To reduce said immunological risks, patients are excluded if they harbor anti-AAV antibodies or have received gene therapy previously. Studies have incorporated immunomodulating or suppressive regimens to block cellular and humoral immune responses such as systemic corticosteroids pre- and post-administration of Luxturna® and Zolgensma®, the two rAAV products with licensed regulatory approval in Europe and the United States. In this review, we will introduce the current pharmacological strategies to immunosuppress or immunomodulate the host immune response to rAAV gene therapy.

Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana.

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50-72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana.

Micro-laminin gene therapy can function as an inhibitor of muscle disease in the dyW mouse model of MDC1A

Gene replacement for laminin-α2-deficient congenital muscular dystrophy 1A (MDC1A) is currently not possible using a single adeno-associated virus (AAV) vector due to the large size of the LAMA2 gene. LAMA2 encodes laminin-α2, a subunit of the trimeric laminin-211 extracellular matrix (ECM) protein that is the predominant laminin expressed in skeletal muscle. LAMA2 expression stabilizes skeletal muscle, in part by binding membrane receptors via its five globular (G) domains. We created a small, AAV-deliverable, micro-laminin gene therapy that expresses these G1–5 domains, LAMA2(G1–5), to test their therapeutic efficacy in the dyW mouse model for MDC1A. We also fused the heparin-binding (HB) domain from HB epidermal growth factor-like growth factor (HB-EGF) to LAMA2(G1–5) to test whether this would increase muscle ECM expression. dyW mice treated intravenously with rAAV9.CMV.HB-LAMA2(G1–5) showed increased muscle ECM expression of transgenic protein relative to mice treated with rAAV9.CMV.LAMA2(G1–5) and showed improved weight-normalized forelimb grip strength relative to untreated dyW mice. Additionally, dyW muscle fibers expressing either micro-laminin protein showed some measures of reduced pathology, although levels of muscle cell apoptosis and inflammation were not decreased. Although systemic expression of rAAV9.CMV.HB-LAMA2(G1–5) did not inhibit all disease phenotypes, these studies demonstrate the feasibility of using a micro-laminin gene therapy strategy to deliver gene replacement for MDC1A.

Abstract PR008: Armed Myxoma virus demonstrates therapeutic activity in pre-clinical xenograft models

Abstract Oncolytic Viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system. This represents a promising therapeutic option for cancer patients that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Poxviridae family of double stranded DNA viruses. The natural host of MYXV is a subset of lagomorphs, but MYXV is able to infect cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins making it an excellent oncolytic virus for introduction of immunomodulatory proteins. The current work describes the oncolytic activity, transgene production capability, and in vivo activity of armed myxoma viruses in human cancer models. This abstract is also being presented as PO090. Citation Format: Lino E. Torres-Dominguez, Lina S. Franco, Mario Abrantes, Benjamin S. Walker, Zachary Tacner, Cassandra Kien, Anna K. Waters, Grant McFadden, Steven J. Potts, Leslie L. Sharp. Armed Myxoma virus demonstrates therapeutic activity in pre-clinical xenograft models [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR008.

Development of the high-productivity marine microalga, Picochlorum renovo, as a photosynthetic protein secretion platform

Microalgae are remarkable in their ability to convert CO2 and sunlight into renewable biomass and bioproducts. Here, we have established a novel photosynthetic protein production platform via computational prediction and genetic engineering using native microalgal protein secretory signal peptides to achieve functional secretion of the fluorescent protein mCherry. Importantly, this work was conducted in the recently characterized alga Picochlorum renovo, an alga of industrial interest due to its rapid growth rate, tolerance to both high temperature and salinity, and genetically tractable nuclear and chloroplast genomes. Genomic queries allowed the identification of native secretory signal peptides, which were N-terminally fused to mCherry allowing for secretion into the culture supernatant. Further characterization revealed no impact on fitness, a production rate of 0.19 mg/L/day, and titer of 0.37 mg/L of transgenic mCherry protein in culture supernatant. These findings lay the foundation for applied genetic engineering approaches that could enable CO2-sequestering, sustainable photoproduction of industrially relevant enzymes at low cost.

Neuroprotective Studies on Polygonum hydropiper L. Essential Oils Using Transgenic Animal Models.

Polygonum hydropiper L. and related species are reported to possess neuroprotective potentials. In an attempt to validate its anti-Alzheimer’s potentials, leaf oils (Ph. Lo) were extensively evaluated in this study against several in vitro and in vivo models of Alzheimer’s disease. The Ph. Lo were tested against pathological targets of Alzheimer’s diseases (ADs). The in vitro and in vivo assays were done for cholinesterase inhibition, anti-radical properties and cognitive assessments using transgenic animal models. In preliminary cholinesterase inhibition assays, Ph. Lo were more active against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS), and hydrogen peroxide (H2O2) radicals. Subsequently, Ph. Lo was evaluated for its effects on special memory, exploratory behavior, and coordination using shallow water maze (SWM), Y-maze, open filed, and balance beam tests. Animal pre-genotyping was done via polymerase chain reaction (PCR) to confirm amyloid precursor protein (APP) transgene, and after completion of drug therapy, brain homogenates from the cortex and hippocampus were evaluated for cholinesterase and free radical studies. In SWM task, disease control animals treated with 10 mg/kg of Ph. Lo for 5 days exhibited significant improvement in cognitive performance indicated by low escape times on 5th day compared with normal animals. In the Y-maze test, transgenic animals showed higher spontaneous alternation behavior than disease control animals and standard control group animals. Ph. Lo therapy has improved the exploratory behavior and declined anxiety behavior in diseased animals as accessed via open field test. Ph. Lo administration significantly augmented the motor and coordination abilities of transgenic animals when compared to other groups of animals and declined AChE, BChE activities as well as free radicals load in the cortex and hippocampus tissues. Based on our finding, it is concluded that Ph. Lo exhibit significant neuroprotective potentials preliminary due to their anti-radicals and cholinesterase inhibitory activities. Ph. Lo need further detailed studies as potential aromatherapy against neurodegenerative disorders.

Expression pattern of the V5-Ostm1 protein in bacterial artificial chromosome transgenic mice.

Mutations in the osteopetrotic transmembrane protein 1 (Ostm1) gene are responsible for the most severe form of autosomal recessive osteopetrosis both in humans and in the gray lethal (gl/gl) mouse. This defect leads to increased bone mass with bone marrow occlusion and hematopoietic defects. To establish the expression profile of the mouse Ostm1 protein in vivo, homologous recombination in bacteria was designed to generate a V5-Ostm1 bacterial artificial chromosome (BAC) that was subsequently integrated in the mouse genome. Tissue expression of the transgene V5-Ostm1 RNA and protein in transgenic mice follow the endogenous expression profile. Immunohistochemistry analysis demonstrated expression in neuronal populations from central and peripheral nervous system and defined a unique cellular expression pattern. Importantly, together with appropriate protein post-translational modification, in vivo rescue of the osteopetrotic bone gl/gl phenotype in BAC V5-Ostm1 gl/gl mice is consistent with the expression of a fully functional and active protein. These mice represent a unique tool to unravel novel Ostm1 functions in individual tissue and neuronal cell populations and the V5-Ostm1 transgene represents an easy visual marker to monitor the expression of Ostm1 in vitro and in vivo.

Digestive fate of transgenic DNA and protein in livestock tissues fed genetically modified feed ingredients: A review

The digestive fate of ingested transgenic DNA and/or proteins in livestock fed a diet containing genetically modified (GM) feed is at present the topic of scientific discussion. A number of studies have investigated the possible transfer and accumulation of novel DNA and/or proteins from transgenic plants in tissues samples of various livestock species, but the data reported are sometimes controversial. Therefore, this study aimed to evaluate the potential transfer and accumulation of transgenic DNA/ proteins from transgenic feed ingredients to livestock through reviewing scientific publications on experimental studies in large and small ruminants, monogastric and fish. Most of the studied experiments were conducted in Europe, and in majority these experiments, transgenic maize or soybean, or both, expressing insect resistant or herbicide tolerant or combination of both, were fed to the experimental animals. More than 50% of the experimental studies evaluated, reported the detection of transgenic DNA/protein from a number of analyzed samples of various livestock species. Transgene detection either in form of DNA or protein was more prevalence in the experimental studies when monogastric animals or fish were used as the experimental animals. Although, these transgenes were also detected in the tissues, organs and products of large and small ruminants, yet the detection frequency was less when compare with that in non-ruminants and fish. Thus overall, findings from this study indicated that transgenic DNA is not completely degraded during feed processing and digestion and that DNA fragments could be detected by well-established detection methods such as PCR or qPCR in a number of organs and tissues of livestock animals.

RNA-based pesticides aim to get around resistance problems

The half-inch-long corn rootworm larva packs a surprising punch. It feasts on the root system of corn before pupating into a black and yellow beetle that does further damage to the plant’s leaves. Before the advent of genetically modified (GM) crops that produce insecticidal proteins to fight rootworm, these insects cost US farmers an estimated $1 billion annually in damage and control measures. As destructive agricultural pests such as the corn rootworm evolve resistance to conventional pesticides, researchers and farmers are looking to RNAi-based treatments as a promising possible alternative. Image credit: Science Source/USDA/Nature Source. But as insects such as the corn rootworm evolve resistance to the suite of traits baked into commodity crops, scientists are queuing up a new application for a biotech tool that targets the protein-making machinery of insects. First identified in 1990, RNA interference (RNAi) entails using double-stranded RNA (dsRNA) to block messenger RNA from its usual function (i.e., sending out instructions to make proteins). With impressive specificity, RNAi can potentially block nucleotide sequences that are only found in a target pest and not in friendly insects or humans. As a result, some scientists are keen on making RNAi the next big tool in agricultural science. The EPA first approved an RNAi pesticide in 2017. That product, called SmartStax Pro, is a GM corn seed that will deploy both transgenic insecticidal proteins and RNAi to fight western and northern corn rootworm. It’s expected to be released in the United States in the next few years (1), according to its maker, Bayer AG, which is headquartered in Leverkusen, Germany. But GM crops are just one of many agriculture-related applications for RNAi. As the cost of producing dsRNA has dropped precipitously, biotech companies are developing dsRNA formulations that could also serve as spray pesticides, making the technology more …

Knocking out SOBIR1 in Nicotiana benthamiana abolishes functionality of transgenic receptor-like protein Cf-4.

Knocking out SOBIR1 in Nicotiana benthamiana by CRISPR/Cas9, abolishes the functionality of the transgenic receptor-like protein Cf-4, recognizing the Avr4 effector of the fungus Cladosporium fulvum.

Effects of genetically modified maize expressing Cry1Ab and EPSPS proteins on Japanese quail

A 49-d feeding study was conducted to evaluate the effects of the genetically modified (GM) maize strain C0030.3.5 on Japanese quails (Coturnix japonica) in terms of body performance and egg quality. Furthermore, the bodily fats of transgenic proteins in the Japanese quails were investigated. The results showed that the parameters body weight, hematology, serum chemistry, relative organ weight, and histopathological appearance were normal in male and female quails that consumed GM diets, and no differences could be attributed to the varying diets in regard to the laying performances or nutrient egg compositions between the groups. Furthermore, the transgenic Cry1Ab and EPSPS proteins were undetectable by Western blot in the blood, organ, fecal, and whole egg samples of quails fed a diet containing GM maize. The results obtained after 49 d suggested that consumption of C0030.3.5 transgenic feed did not adversely affect quail health or egg quality, and there was no evidence of transgenic protein translocation to the blood, tissues, feces, and eggs. Based on the different parameters assessed, C0030.3.5 transgenic maize is a safe food source for quails that does not differ in quality from non-GM maize.