Transgenic Research最新文献

Deficiency of the Monocarboxylate Transporter 8 (MCT8) severely impairs thyroid hormone (TH) transport into the brain, disrupting brain development as well as peripheral TH homeostasis. Studies assessing MCT8 expression patterns and tissue-specific pathologies induced by local TH-deficiency are often inconclusive due to unreliable antibody staining and the lack of functional tools to specifically target MCT8-expressing cells. For this purpose, we generated non-inducible Mct8-Cre and tamoxifen-inducible Mct8-CreERT2 mice. Mct8-Cre;Sun1-sfGFP mice demonstrated ubiquitous Sun1-sfGFP expression, due to early recombination driven by Mct8 gene expression at the stage of trophoblast implantation. Tamoxifen injection in 6-week-old Mct8-CreERT2 mice induced reporter expression specifically in Mct8-expressing cells in the brain and peripherally in liver, kidney, and thyroid, without leaky reporter expression in vehicle controls. Using vDISCO tissue clearing and 3D-imaging of GFP-nanobody-boosted mice, we further identified the sublingual salivary gland and the prostate as prominent Mct8-expressing organs. Nuclei from Mct8-expressing cells in the brain could selectively be enriched using fluorescence-activated nuclei sorting on Mct8-CreERT2;Sun1-sfGFP mice and characterized as choroid plexus cells and tanycytes. Our new inducible Mct8-CreERT2 line provides researchers with a tool to reliably mark, enrich, and characterize Mct8-expressing cells and to genetically modify genes specifically in these cells to study thyroid hormone transport and function.

Advancements in genomic crop techniques have led to the development of new genetic technologies, such as base- and prime editing, but improvements have been made to existing conventional techniques as well. Fields in which these advancements occur include targeted mutagenesis, conventional random mutagenesis, and developments with null segregants, e.g., crops from which transgenic elements have been crossed out. In this review, we describe the developments in these three fields and provide considerations concerning regulatory and safety aspects. Because of differences in legislation of modern biotechnology between countries or regions, regulatory challenges are to be expected given the ongoing developments in genomic crop techniques. Moreover, the nature of the mutations induced with these newly developed techniques is not different from those induced with conventional techniques, making the modified crop plants indistinguishable from non-modified counterparts of the same crop species. Thus, enforcement of regulations cannot solely rely on technical analytical methods. Also, potential off-target or unintended effects in the primary mutants remain underexplored. Yet, these do not raise safety concerns owing to the experience with the crop breeding practice of iterative cycles for desirable traits selection, as well as the segregation and discard of unwanted phenotypes. Given that regulation will always change after innovation and developments within the sector advance rapidly, we advocate that both authorities and the breeding sector pro-actively implement a food safety culture. Such a safety culture will help developers of genomic technologies in crops to identify potential food safety issues at an early stage of development of future products.

Transplastomic technology has found diverse applications in metabolic and resistance engineering, yet its implementation has been primarily limited to select plant species like Nicotiana tabacum. Here we report a robust and reproducible plastid transformation system tailored for Solanum nigrum (black nightshade), a significant vegetable and medicine plant within Solanaceous family. A S. nigrum-specific plastid vector, strategically designed for integration between the trnfM and trnG genes of the plastid genome, harbored the spectinomycin-resistance gene (aadA) as a selectable marker and green fluorescent protein (gfp) as a reporter gene. The vector was delivered via biolistic bombardment into leaf explants of S. nigrum, achieving an overall efficiency of approximately fifteen transplastomic events per shot. The site-specific integration of foreign genes and the establishment of a high homoplastomic state were verified through PCR assays and Southern blot analyses. Confocal laser scanning microscopy confirmed the presence of GFP fluorescence in chloroplasts, with GFP accumulation reaching about 2% of the total soluble protein in leaves. Crossing experiments between transplastomic plants and wild-type plants demonstrated the maternal inheritance of the S. nigrum plastid genome. The successful establishment of S. nigrum transplastomic technology holds promise for fostering novel synthetic biology applications within medicinal plant research.

Transgenesis mediated by transposon is an effective approach for introducing exogenous DNA into the nuclear genome and establishing stable transgenic strains that efficiently express genetic tools. Although the DNA transposon Tol2 is widely used for transgenesis in zebrafish, its endogenous transpositional activity can lead to unintended transgene mobilization, making it unsuitable for transgenesis in medaka (Oryzias latipes). Here, we demonstrated that the DNA transposon Tip100, originally identified in the common morning glory (Ipomoea purpurea), an ornamental plant, can serve as a useful tool for transgenesis in Japanese medaka. The GFP transgene cassette, when co-injected with Tip100 transposase mRNA, was expressed in significantly higher number of somatic cells in the injected fish. Furthermore, a transgene flanked by truncated recognition sequences (100 bp each) exhibited expression levels comparable to those of the original vector containing the full 2.2 kb recognition sequence. Injection of a transgene driven by a germline-specific promoter revealed that fish injected with Tip100 mRNA exhibited a significantly higher germline transmission rate (42/68; 62.7%) compared to those injected without the mRNA (13/62; 21.0%). We successfully established transgenic strains by outcrossing injected founders with GFP-positive germ cells (7/7; 100%) and demonstrated that the transgenes were randomly integrated into the medaka genome, generating 8-bp duplications at the insertional sites-an insertional signature of the hAT superfamily of transposons. Our findings indicate that the Tip100 system is a promising tool for generating stable transgenic strains that express various genetic tools in medaka and potentially other fish species.

Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5' UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5' end of the Cas9 cDNA to function as 5' UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20-21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20-21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.

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.