Transgenic Research最新文献

In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.

The production of transgenic animals using non-viral methods has raised questions regarding their long-term health and genomic stability. In this study, we evaluated these aspects in transgenic cattle over ten years, using transposon-mediated gene transfer. Our longitudinal analysis included a comprehensive health assessment and whole-genome DNA resequencing. We found no significant alterations in physiological parameters or health complications in transposon-mediated transgenic cattle that exceeded 10 years of age. Genomic analysis revealed that the rates of somatic mutations and copy number variations in transgenic cattle were comparable to those in non-transgenic cattle. Furthermore, structural variants were infrequent, suggesting that transposon-mediated gene insertion did not compromise genomic integrity. These findings highlight the viability of transposon systems for generating transgenic livestock, potentially expanding their applications in agriculture and biotechnology. This study contributes significantly to our understanding of the long-term implications of transgenesis in large animals and supports the safety and stability of this method.

Mosquitoes visit flowers to obtain sugar or other nutrients and therefore possibly serve as major or minor pollinators of some plant species. They also often derive plant nutrients from other sources, such as extrafloral nectaries and honeydew. In a few cases, the plant-mosquito relationship is close, and mosquito pollination has been confirmed. Most plant species visited by mosquitoes, however, appear to depend on multiple means of pollination, particularly other flower-feeding insects. In addition, most mosquito species visit the flowers of many kinds of plants, possibly dispersing pollen in both biologically meaningful and irrelevant ways. This apparent lack of selectivity by both plants and mosquitoes liberates each of them from dependence on an unreliable pollen vehicle or nutrient source. A hypothetical pollinating role for the two top vectors of devastating human-disease pathogens, Anopheles gambiae or Aedes aegypti, relies on indirect evidence. So far, this evidence suggests that their participation in pollen transfer of native, introduced, or beneficial plants is negligible. The few plant species likely to be pollinated by these vectors are mostly invasive, harmful weeds associated with humans. That conclusion draws support from four characteristics of these vectors: (1) the numerous alternative potential pollinators of the flowers they visit; (2) their common use of diverse non-floral sources of nutrients; (3) the females' infrequent sugar feeding and heavy reliance on human blood for energy; and (4) their relatively low population densities. From these traits it follows that focused suppression or elimination of these two vectors, by whatever means, is highly unlikely to have adverse effects on pollination in endemic biotic communities or on ornamental plants or food crops.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

Root-specific or preferential promoters are essential to genetically modify plants with beneficial root traits. We have characterised the promoter from an oil palm metallothionein gene (EgMT) and performed a serial 5' deletion analysis to identify the region(s) essential for transgenes expression in roots. Stable functional characterisation of tobacco transgenic lines using the T₁ generation showed that a deletion construct, designated as RSP-2D (1107 bp), directed strong GUS expression at all stages of root development, particularly in mature roots. Other constructs, RSP-2A (2481 bp) and RSP-2C (1639 bp), drove GUS expression in roots with an intensity lower than RSP-2D. The promoter activity was also detectable in seed pods and immature seeds, albeit at lower levels than CaMV35S. The promoter activity may also be induced by wounding as intact GUS staining was observed at the flower- and leaf-cutting sites of T₁ samples carrying either RSP-2C or RSP-2D constructs. The promoter sequence contains cis-acting elements that may act as negative regulators and be responsible for root specificity. The results further indicated that the 5' UTR and ATATT sequences are essential for strong promoter activity. This study highlights the potential of RSP-2D promoter as a tool for modifying root traits through genetic engineering.

We report the generation and characterization of the K5: CAT bigenic mouse in which the constitutively activated form of β-catenin (ΔN89 β-catenin) is conditionally expressed in cytokeratin-5 (K5) positive epidermal keratinocytes. Following short-term doxycycline intake during the telogen resting phase, the adult K5: CAT bigenic develops enlarged pilosebaceous units that expand deep into the dermis, an expansion usually observed during the anagen growth phase. Prolonged doxycycline treatment results in significant thickening and folding of the K5: CAT epidermis. During this persistent induction period, there is clear evidence of increased keratinocyte proliferation, particularly in the epidermal basal cell layer and the outer root sheath of the hair follicle. This unscheduled increase in cellular proliferation likely explains the decrease in hair density observed in the K5: CAT mouse following persistent doxycycline intake. Numerous hyperplastic endometrioid cysts, which display cornification toward their lumens, are also observed during this treatment period. Remarkably, de-induction of ΔN89 β-catenin expression through doxycycline withdrawal results in a marked reversal of the skin phenotype, suggesting that these morphological changes are dependent on continued signaling by β-catenin and/or its downstream molecular mediators. Joining a small group of mouse models for conditional β-catenin signaling, our K5: CAT mouse model will be particularly useful in identifying those molecular mediators of β-catenin that are responsible for initiating and maintaining these phenotypic responses in the K5: CAT skin. Such studies are predicted to shed more light on β-catenin signaling in epidermal epithelial morphogenesis, hair follicle cycling, and hair growth pathologies.

In recent years there have been major advances in precision breeding technologies, such as gene editing, that offer promising solutions to revolutionise global crop production and tackle the pressing issues in food systems. The UK has leading expertise in genomics, and research is already taking place to develop crops with improved resilience to climate change, resistance to disease and less reliance on chemical inputs. In March 2023, the Genetic Technology (Precision Breeding) Act received Royal Assent and passed into UK law. It provides a framework from which to build more proportionate regulations for plants and animals made using genetic technologies which contain genetic changes that could also arise through traditional breeding-known as 'Precision Bred Organisms'. New legislation and the utilization of UK world-leading research could help to enhance the efficiency of breeding systems and enable the development of plants and animals that are healthier, better for the environment and more resilient to climate change.

Lettuce is one of the most widely consumed vegetables in the world, commonly eaten fresh in salads, sandwiches, wraps, and as a garnish in various dishes. Consequently, it is a very promising vehicle to deliver vitamins, such as folate (vitamin B9), to a specific population using biofortified varieties generated by conventional or molecular breeding. A new genetically modified lettuce was generated with increased folate content. However, some issues related to public perception regarding this technology should still be evaluated. The aim of this study was to analyze whether consumers are willing to accept a folate-biofortified GM lettuce that could become available to the Brazilian market. A questionnaire involving several issues regarding lettuce consumption was answered by 2,391 people from almost all Brazilian states. When informed that the folic acid biofortified lettuce is a transgenic plant, 46.1% of respondents stated that they would eat it and 30.5% stated that it would be a possibility. This study demonstrated that if there is any explanation regarding the advantage in relation to the use of biotechnology, like enrichment with folic acid, the number of people who accept it increases.

Monellin is a sweet protein that may be used as a safe and healthy sweetener. However, due to its low stability, the application of monellin is currently very limited. Here, we describe a wild-type, a double-sites mutant (E2N/E23A) and a triple-sites mutant (N14A/E23Q/S76Y) of single-chain monellin (MNEI) expressed in transgenic mice milk. Based on enzyme-linked immunoassay (ELISA), Western blot, and sweetness intensity testing, their sweetness and stability were compared. After boiling for 2 min at different pH conditions (2.5, 5.1, 6.8, and 8.2), N14A/E23Q/S76Y-MNEI showed significantly higher sweetness and stability than the wild-type and E2N/E23A-MNEI. These results suggest that N14A/E23Q/S76Y-MNEI shows remarkable potential as a sweetener in the future.