Transgenic Research最新文献

Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both homeostatic and pathological processes. Human CLPs such as YKL-40 have been proposed as biomarkers of disease severity in many conditions. Murine CLPs Brp39, Ym1, and Ym2 are similarly upregulated in multiple mouse models of pathology. Investigation of Ym1 and Ym2 is hampered by recent gene duplication events on the C57BL/6, but not BALB/c, background leading to complexity in the genomic locus. Here, we have generated a Ym1 deficient mouse using a novel CRISPR-Cas9 targeting approach involving CB6 (C57BL/6 X BALB/c) mixed background embryos. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein. Additionally, expression of related genes including Chia, Chil1, and Chil4 were not altered in Ym1-deficent animals. This new transgenic mouse line will be key for future investigations of CLP functions and the utilised approach to genetic manipulation may provide a useful strategy for other genes which show differences in copy number between inbred mouse strains.

The major histocompatibility complex class I (MHC I) is crucial in adaptive immunity, enabling CD8 + T cells to detect and eliminate infected and cancerous cells. Recent studies have uncovered significant variability in MHC I expression across tissues, challenging the traditional belief of uniform expression. Lung epithelial cells (LECs) express meager amounts of MHC I, which preserves the lung epithelium from excessive inflammation but renders it more susceptible to cancer and infection. Despite MHC I overexpression in various immunopathologies, its precise role in disease initiation or progression remains unclear due to the absence of suitable in vivo models for studying MHC I overexpression. This study introduces a novel mouse model with targeted surface MHC I upregulation. Leveraging a conditional Cre-lox system, we augmented Nlrc5 expression to specifically upregulate MHC I in alveolar type 2 (AT2) LECs, known for their low basal expression of MHC I and significant overexpression in disease. Our model demonstrated a rapid and sustained tenfold increase in MHC I surface expression persisting for up to a year without triggering pathology or inflammation. Comprehensive characterization and validation of this model indicated that MHC I overexpression does not serve as a primary initiator of respiratory diseases under steady-state conditions and shows a therapeutic window for increasing MHC I without significant damage to the lung epithelium. This adaptable model offers insights into the effects of tissue-specific MHC I regulation and presents new avenues for therapeutic development.

An ecological risk assessment (ERA) was conducted for MON 89151, which expresses three proteins (Cry1Da_7, Cry1B.3, and Vip3Cb1) developed to help protect against lepidopteran pests such as Heliothis virescens, Helicoverpa zea, and Spodoptera frugiperda. The ERA focused on evaluating the potential risks to beneficial non-target organisms (NTOs) from MON 89151 cultivation, by examining the protein's mode of action, insecticidal activity spectrum, ecological exposure levels, potential for environmental persistence, and hazard to representative NTO taxa under laboratory conditions. The protection goal driving the ERA was preserving key ecosystem services provided by NTOs in agriculture. The Cry1Da_7 and Cry1B.3 proteins, the same and/or similar to previously registered (Cry1Da_7 in MON 95379 maize) and/or reviewed (Cry1B.2 in MON 94637 soybean) insecticidal proteins, have been demonstrated to pose negligible risks to NTOs, enabling a bridging approach to existing hazard testing for these proteins. The third protein in MON 89151, Vip3Cb1 also demonstrated no adverse effects on NTOs at or above expected environmental concentrations under laboratory conditions. Therefore, the ERA concluded that cultivation of MON 89151 would pose minimal ecological risk to NTOs, supporting its safety in agricultural ecosystems.

Brinjal (eggplant) plays a crucial role in income generation for smallholder farmers but faces severe yield losses due to the Eggplant Fruit and Shoot Borer. Since 2014, an insect-resistant genetically modified brinjal (Bt brinjal) has been available in Bangladesh, yet its adoption remains low, with many farmers discontinuing its use. To better understand the implementation of GM crops beyond initial adoption, this study examines the factors influencing farmers' intention to continue adopting Bt brinjal. Using an extended expectation confirmation model, this study investigates how pre-adoption factors, such as performance and effort expectancies, influence the (dis)confirmation of expectations and how post-adoption factors, including perceived usefulness and satisfaction, shape farmers' intention to continue Bt brinjal adoption. Based on a structured survey with 151 Bt brinjal adopters, the proposed model was tested using partial least squares structural equation modeling (PLS-SEM). Results reveal that pre-adoption performance and effort expectancies played a crucial role in shaping confirmation, which subsequently affected post-adoption perceived usefulness and satisfaction. Furthermore, satisfaction and perceived usefulness were key drivers of farmers' intentions to continue adopting Bt brinjal. The study contributes to the literature by integrating pre- and post-adoption constructs to explain continuance behavior and provides actionable insights for policymakers and stakeholders to enhance farmer satisfaction and long-term adoption of GM food crops.

The global food system has inadequately addressed complex societal challenges, including climate change and nutritional deficiencies. There is an increasing recognition of the interconnectedness of human and planetary health in food production and consumption. Several policy interventions exist to tackle food-related nutritional and environmental aspects and influence consumer decision-making towards nutritious and environmentally friendly options. Examples of demand-side interventions include food-based dietary guidelines (FBDGs), product labels, and taxes. In this contribution, a discussion of recent developments in demand-side interventions and their potential shortcomings highlights the need for different solutions. Advancing personalised dietary advice is an alternative to FBDGs and complementary to existing interventions, requiring a conceptual shift away from generic and population-based suggestions. Clustering and classification analysis of actual individual dietary patterns into citizen profiles is the first key step to developing relatable advice for each profile. For a transition toward plant-rich diets, this approach recognises (a) differentiated nutritional needs and deficiencies and (b) profile-sensitive 'starting points' for environmental impact reduction. In essence, while lowering the environmental impact, variability is incorporated at two levels: food choices among citizen profiles, obtained from the clustering and classification analysis, and nutritional adequacy for each profile. A first set of such citizen profiles has been reported for the Belgian population. We recommend further application of this approach in populations in other geographies. The next key steps towards developing relatable advice for the obtained citizen profiles demand that we start looking at the cultural consumption habits and strive for improved food literacy in our societies.

Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play pivotal roles in glucose homeostasis and metabolic regulation. Therapeutic incretin receptor agonists (RAs), such as tirzepatide, are widely used to manage type 2 diabetes and obesity. However, incretin RAs are facing production challenges at present. Therefore, we engineered transgenic (tg) mice to secrete incretin RAs in milk, leveraging mammary gland bioreactors for cost-effective peptide production. The goat beta-casein promoter-driven constructs encoding tirzepatide-derived peptide linked to human IgG4 Fc via a (GGGGS)₃ spacer were used to produce tg mice. Founders tg-1 and tg-5 exhibited mammary-specific expression, yielding 0.8-1.42 g/l recombinant protein exclusively in milk. Progeny nursed by founders showed sustained hypoglycemia (10-39% reduction; p < 0.05) and marked weight loss (14-49%; p < 0.01) compared to wild-type controls, validating the bioactivity of milk-derived GLP-1/GIP RAs. Moreover, tg-5-nursed offspring experienced high mortality post-Day 16, likely due to overdosing. This proof-of-concept demonstrates the mammary gland bioreactor as a viable platform for incretin RAs production, circumventing complex synthesis and enabling scalable biologics manufacturing.

A new line of cedar pollen allergen-accumulating transgenic rice, which accumulated the 7Crp peptide comprised of 7 concatenated major T cell epitopes of cedar pollen allergens, was developed; it improved on the shortcomings of an original transgenic line. The new line has one copy of inserted T-DNA and a substantial expression of the 7Crp peptide, and was named Os7Crp2. Regulatory approval is needed before commercializing this cedar pollen peptide rice as a genetically modified food product in Japan. Therefore Os7Crp2 was evaluated for the criteria required for food safety assessments, under the guidance of Standards for the Safety Assessment of Genetically Modified Foods (Seed Plants) in Japan. No statistically significant differences were found between Os7Crp2 and the control (parental) Dongtokoi cultivar in physicochemical properties, expressed traits, or concentrations of key components, except for the insertion of the T-DNA and expression of the recombinant protein derived from the transgenes. These results suggest that Os7Crp2 is compositionally equivalent to non-transgenic rice.

Molecular breeding and gene function studies in plants require high transformation efficiency. Agrobacterium-mediated transformation has contributed significantly to molecular research in many plants, but is inefficient and inconsistent in rice that do not host Agrobacterium. Transformation efficiency in rice remains low. Therefore, this study aimed to establish a simple and efficient transformation method for rice using Agrobacterium. Two foreign genes (CISP1-GFP and CISP2-GFP) and Agrobacterium strain (EHA105) was used in the experiments. Then, Agrobacterium infection of rice seeds that had absorbed water and germinated under reduced pressure infiltration conditions showed that an average of 14% of the seeds formed after growth (12% with CISP1-GFP and 16% with CISP2-GFP) carried the foreign gene, and it was also confirmed by PCR, Western blot, GFP fluorescence and TAIL-PCR. Since this method does not involve callus formation or re-differentiation of rice plants, no special equipment or complicated operations are required, and transformants can be obtained in only three months. Therefore, this method is expected to simplify rice genetic manipulation and promote molecular breeding of rice.

Immunodeficient mouse models are invaluable tools for preclinical research, particularly for cancer therapies and studies of the human immune system. Notably, strains with combined Prkdc (scid) and Il2rg (null) mutations-such as NOG and NSG mice- are widely used due to their profound immunodeficiency, allowing efficient engraftment of various human cells. However, these models were generated by disrupting the Il2rg gene through replacement with a neomycin resistance (Neo) cassette in embryonic stem cells. Incomplete excision of this cassette can inadvertently alter the expression of neighboring genes, thereby introducing potential confounding variables. In addition, they may still express mutant mRNAs that escape nonsense-mediated decay (NMD) and/or produce truncated proteins with residual activity, potentially compromising the interpretation of experimental outcomes. To address this, we developed the N2G mouse strain (NOD-2-Genes KO) where almost all genomic loci of both Prkdc and Il2rg genes are deleted via CRISPR/Cas9 genome editing. N2G mice exhibited tumor growth comparable to NOG mice following the transplantation with several human cancer cell lines. Moreover, human CD34⁺ cord blood (CB) cells engrafted into N2G mice showed robust reconstitution of human immune cells, especially T cells in peripheral blood, spleen and bone marrow, compared to NSG mice. These results suggest that N2G mice, lacking residual mutant mRNA and the exogenous Neo resistant gene, offer an advanced model for preclinical studies.