Transgenic Research最新文献

Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.

The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b^dl40, keap1a^dl07, and nfe2l2a^dl703 (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H₂O₂-induced oxidative stress, keap1b^dl40 larvae, unlike their keap1a^dl07 counterparts, exhibited extreme sensitivity to the lethal effects of CuSO₄ exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b^dl40 null allele) for future studies.

Developing climate-resilient plants with enhanced tolerance to abiotic stresses such as drought and salinity is essential for mitigating the impact of climate change on agriculture. One promising approach is the heterologous expression of stress-responsive genes under appropriate regulatory elements. In this study, we evaluated the potential of the CsATG6 gene from cucumber (Cucumis sativus) to improve abiotic stress tolerance through heterologous expression and examined its promoter's utility for driving transgene expression in dicots. Overexpression of CsATG6 in Arabidopsis enhanced seedling root growth under nitrogen deficiency, drought, and salt stress, and improved survival rates following high-salinity treatment, indicating its potential application in stress-tolerant crop development. The CsATG6 promoter was cloned and analyzed for cis-acting elements responsive to environmental stimuli, hormones, and developmental signals. A series of 5' deletion constructs were fused to a GUS reporter and tested in Arabidopsis, tobacco leaves, and cassava calli under various treatments, including salicylic acid (SA), abscisic acid (ABA), drought, salt, and nitrogen starvation. Among the tested constructs, pCsATG6::GUS-1027 and pCsATG6::GUS-512 showed strong and inducible expression in response to ABA, SA, and multiple abiotic stresses. Furthermore, an ABA-repressive element within the promoter was identified and functionally validated via site-directed mutagenesis. Together, these findings demonstrate that CsATG6 is a valuable candidate gene for enhancing stress tolerance through heterologous expression, and that its promoter fragments can serve as stress- and hormone-responsive regulatory elements for transgene expression in some dicots, including economically important crops like cassava.

Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.

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.