Transgenic Research最新文献

{"title":"Highly efficient direct seed transformation protocol for japonica rice (Oryza sativa L.) by Agrobacterium tumefaciens: overcoming the complexity of callus regeneration and avoiding the occurrence of somaclonal-mutations.","authors":"Md Maksudul Haque, Yutaro Okumura, Shin-Ichiro Kidou","doi":"10.1007/s11248-025-00453-w","DOIUrl":"10.1007/s11248-025-00453-w","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"36"},"PeriodicalIF":2.0,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144733450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of NOD SCID mice with near-complete deletions of Il2rg and Prkdc for human cancer and HSC engraftment.","authors":"You-Min Kim, Hee Ju Na, Do Hee Kwon, Jae Hoon Lee, Bo Min Park, Subin Lee, Tae Wook Nam, Mi Yeon Park, Sun Ha Park, Sung Joo Kim, Bongkum Choi, Han-Woong Lee","doi":"10.1007/s11248-025-00454-9","DOIUrl":"10.1007/s11248-025-00454-9","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"35"},"PeriodicalIF":2.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254168/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144609630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.","authors":"Hiralben Lakhani, Naveen Kumar, Alka Jangra, Sanjana Negi, Thobhanbhai Dholariya, Siddharth Tiwari","doi":"10.1007/s11248-025-00451-y","DOIUrl":"https://doi.org/10.1007/s11248-025-00451-y","url":null,"abstract":"","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"34"},"PeriodicalIF":2.7,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144592436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Competent expression of effective and long-acting human α-Gal A-Fc fusion protein in the milk of transgenic mice.","authors":"Mengke Yuan, Lan Yang, Chunyang Li, Zhiqiang Wu, Zhipeng Liu, Tianfei Du, Xinzong Rong, Cong Cong, Yongxia Zhang, Xiaoping Yu, Yali Gao, Zhengli Chen, Lanjun Liu, Yonghong Ge","doi":"10.1007/s11248-025-00452-x","DOIUrl":"https://doi.org/10.1007/s11248-025-00452-x","url":null,"abstract":"<p><p>Fabry disease is a rare X-linked inherited lysosomal storage disorder caused by a reduction or deficiency in the activity of α-galactosidase A (α-Gal A). The short half-life of α-Gal A necessitates biweekly infusions, thereby imposing significant economic and physical burdens on patients and their families. In this study, a novel long-acting replacement for α-Gal A, termed α-galactosidase A-Fc (α-Gal A-Fc), was designed. Two transgenic founders with an 18.2% transgene rate were obtained to express recombinant human α-Gal A-Fc protein in mouse milk. The α-Gal A-Fc enzyme activity in the milk of high-copy mice were significantly higher than those in low-copy mice and were stably inherited across F1-F3 generations. No significant differences were observed in α-Gal A-Fc concentration or enzymatic activity among high-copy mice of the same generation. During early lactation, the α-Gal A-Fc concentration and enzymatic activity were 2.1-fold and 2.17-fold higher, respectively, compared to late lactation. The expression levels during late lactation did not affect purification efficiency, allowing for the pooling of milk from high-copy mice throughout the entire lactation period for protein purification. The elimination half-life of the purified α-Gal A-Fc protein in mouse serum was 471 min, approximately 43 times longer than that of the commercially available drug Replagal. These findings facilitate the development of an efficient production system for long-acting human α-Gal A-Fc fusion protein and provide valuable insights into the utilization of transgenic large animal mammary gland bioreactors for biopharmaceuticals.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"33"},"PeriodicalIF":2.7,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144544984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Omega-3 polyunsaturated fatty acids ameliorate high-fat-diet-induced structural and functional impairments of testicular tissue via ER stress inhibition.","authors":"Jiaxi Ju, Shuangli Wen, Xuan Zhao, Jiyuan Cheng, Hongjin Yang, Guiming Zhu","doi":"10.1007/s11248-025-00448-7","DOIUrl":"10.1007/s11248-025-00448-7","url":null,"abstract":"<p><p>Obesity is a well-established risk factor for male infertility. Recent studies have demonstrated that endoplasmic reticulum (ER) stress is a key contributor to spermatogenic disorder associated with obesity. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to mitigate ER stress, thereby alleviating insulin resistance. However, their specific role in obesity-induced reproductive disorders remains unclear. In this study, we used the transgenic fat-1 mice (TG mice) that are capable of endogenously converting Omega-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. The mice were divided into four groups according to their diet: a control group (WT + ND, n = 8), a wild type high-fat diet group (WT + HFD, n = 8), a transgenic control group (TG + ND, n = 8), and a transgenic high-fat diet group (TG + HFD, n = 8). After 18 weeks of feeding, the mice were anesthetized and euthanized to examine indicators related to obesity and reproductive function. High-fat diet (HFD) induced significant obesity in WT mice, and we observed significant alteration mitophagy in the reproductive function of WT mice (P < 0.001), primarily manifested as abnormal testicular morphology, decreased sperm quantity and motility (P < 0.01), and reduced testosterone levels (P < 0.01). TG mice exhibited a significant attenuation of these pathological changes (P < 0.05). Markers of ER stress and mitophagy were significantly reduced in the testes of TG mice (P < 0.01), accompanied by an increased expression of phosphorylated AMP-activated protein kinase (AMPK) (P < 0.01), compared to WT mice. Concurrently, TG mice exhibited significantly elevated levels of mitochondrial biogenesis markers and key enzymes involved in testosterone synthesis in the testes, compared to those in WT mice (P < 0.01). Furthermore, TG mice displayed notable resistance to testicular inflammation induced by HFD compared with WT mice (P < 0.01). Our findings suggest that HFD-induced obesity is associated with impaired testicular morphology and function in mice. n-3 PUFAs may ameliorate these impairments by activating AMPK to suppress ER stress, restore mitochondrial dysfunction, and alleviate inflammation, thereby improving testicular morphology and function.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"32"},"PeriodicalIF":2.7,"publicationDate":"2025-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Agrobacterium-mediated genetic transformation of chia (Salvia hispanica L.), a rich source of omega-3 fatty acid.","authors":"N Anirudh, P K Haritha, R V Sreedhar","doi":"10.1007/s11248-025-00450-z","DOIUrl":"https://doi.org/10.1007/s11248-025-00450-z","url":null,"abstract":"<p><p>Chia (Salvia hispanica L.), a nutritionally valuable crop, is the richest source of α-linolenic acid, a key omega-3 fatty acid. Despite its nutritional benefits, a stable genetic transformation method for chia is not available. This study presents a sonication-assisted Agrobacterium-mediated transformation protocol optimized for high-efficiency transformation of chia seedlings. Key parameters including bacterial cell density, acetosyringone concentration, sonication duration, vacuum infiltration, and infection time were optimized. Results demonstrated that an optical density at 600 nm (OD₆₀₀) of 0.5, acetosyringone concentration of 100 µM, 20 min of sonication, 10 min of vacuum infiltration, and 60 min of infection significantly enhanced transformation efficiency and GUS expression. This optimized protocol was validated through Polymerase Chain Reaction and β-glucuronidase (GUS) assay in transformed plants. Our findings establish a robust and reliable transformation protocol, paving the way for future genetic engineering efforts aimed at enhancing the nutritional and agronomic traits of chia.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"31"},"PeriodicalIF":2.7,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144326905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression of an antimicrobial peptide persulcatusin fused with calmodulin in rice cultured cells.","authors":"Gaku Fujita, So Shimoda, Minako Itagaki, Takuto Yahara, Ryuta Tobe, Hiroshi Yoneyama, Yukihiro Ito","doi":"10.1007/s11248-025-00449-6","DOIUrl":"10.1007/s11248-025-00449-6","url":null,"abstract":"<p><p>Antimicrobial proteins and peptides are potential alternatives to antibiotics. Persulcatusin is an antimicrobial peptide found in the taiga tick Ixodes persulcatus. We constructed fusion genes that encode, from the N-terminus to the C-terminus, a signal sequence of rice α-amylase 3D, mouse calmodulin, a target sequence of TEV protease of tobacco etch virus, and persulcatusin with or without a His tag at the N-terminus of the mature fusion protein. These fusion genes were then introduced into rice. Western blot analysis detected persulcatusin fusion proteins in transgenic calli, suspension cells, and their culture medium. Antimicrobial activity against Staphylococcus aureus was detected in the protein extracts prepared from the transgenic callus but not from the non-transgenic wild-type callus, and TEV protease treatment to release persulcatusin from the fusion protein enhanced antimicrobial activity. The growth of the transgenic rice plants was unaffected. Our results indicate that functional persulcatusin can be produced in rice cells. This provides a basis for the mass production of persulcatusin for therapeutic use against bacterial infectious diseases in humans and livestock.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"30"},"PeriodicalIF":2.7,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12170776/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour.","authors":"Ekta Patel, Piyali Das, Somak Hazra, Manveer Sharma, Gautam Chhabra, Balwinder Singh Gill, Sucheta Sharma, Ajinder Kaur, Deepak Singla, Jagdeep Singh Sandhu","doi":"10.1007/s11248-025-00447-8","DOIUrl":"10.1007/s11248-025-00447-8","url":null,"abstract":"<p><p>Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T₀ putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T₁ and T₂ seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T₁ progeny; segregation analysis revealed homozygosity and heritability of mutation in T₂ plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T₂ seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"29"},"PeriodicalIF":2.7,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12158852/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144267291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.","authors":"Hiralben Lakhani, Naveen Kumar, Alka Jangra, Sanjana Negi, Thobhanbhai Dholariya, Siddharth Tiwari","doi":"10.1007/s11248-025-00446-9","DOIUrl":"10.1007/s11248-025-00446-9","url":null,"abstract":"<p><p>The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"28"},"PeriodicalIF":2.7,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144209615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"First report of transgenic Agave sisalana 'RLV 19' plants resistant to the herbicide glyphosate-an improved alternative for biofuel production.","authors":"Carolina Rossi de Oliveira, Aline Vitória Corim Marim, Camila Santos Woloche, Paulo Cezar De Lucca, Marcelo Falsarella Carazzolle, Gonçalo Amarante Guimarães Pereira","doi":"10.1007/s11248-025-00445-w","DOIUrl":"https://doi.org/10.1007/s11248-025-00445-w","url":null,"abstract":"<p><p>The Agave genus is recognized for its diversity, economic importance, and adaptability to arid and semi-desert climates. Among its species, Agave sisalana stands out for the production of sisal, a resilient natural fiber with various industrial applications and potential to be used as a raw material in biofuel production, due to the accumulation of fermentable sugars in its biomass. Although this species presents significant agronomic potential, challenges in conventional breeding hinder its cultivation. A viable alternative for genetic improvement is the development of transgenic plants that incorporate desirable agronomic traits through the controlled insertion of genes of interest into their genome. The objective of study was to develop a protocol for obtaining transgenic plants of Agave sisalana 'RLV19,' a species widely cultivated in the semi-arid region of Bahia-BA, Brazil, using the cp4-epsps gene aimed at constitutive gene expression. Two transgenic plants of Agave sisalana 'RLV19' were regenerated via organogenesis. PCR analyses and CP4-EPSP protein expression by RT-PCR confirmed the presence and expression of the transgene in these plants. This is the first report of A. sisalana transgenic plants expressing the cp4-epsps gene, and to the best of our knowledge, there have been no prior reports on protocols for the production of transgenic plants of this species.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"27"},"PeriodicalIF":2.7,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144209614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}