Lídia N Queiroz, Tatiane R Monteiro, Ana C M M Gomes, Glaucia B Cabral, Francisco J L Aragão
{"title":"Enhanced folate content in bioengineered lettuce stably expressed under field conditions.","authors":"Lídia N Queiroz, Tatiane R Monteiro, Ana C M M Gomes, Glaucia B Cabral, Francisco J L Aragão","doi":"10.1007/s11248-025-00440-1","DOIUrl":"10.1007/s11248-025-00440-1","url":null,"abstract":"<p><p>Folate (vitamin B9) is an essential nutrient that plays a crucial role in various bodily functions. Its deficiency can lead to health issues, such as megaloblastic anemia, neural tube defects in the developing fetus and increased risk of cardiovascular diseases. Consequently, there is interest in increasing folate content in food crops by classical and molecular breeding. Since folate is a water-soluble vitamin that is sensitive to heat, we chose to manipulate its synthesis pathway in lettuce, which is often used for salads and sandwiches, without heat treatment, which makes it a good vehicle for making folate available to the population via biofortification. Transgenic lettuce plants were generated to express the GTP-cyclohydrolase I and aminodeoxychorismate synthase coding sequences from Arabidopsis thaliana. Plants were cultivated under greenhouse conditions and two field trials were carried out. Results have shown that transgenic lines presented up to 3.4 times more folate than the non-transgenic lettuce plants and 1.9 times more folate than spinach, considered one of the plants richest in folates. Advanced generations of homozygous plants were cultivated under field conditions for two years and the results showed that folate-biofortification was stable. A folate-biofortified lettuce serving would provide about 36 to 64% of the recommended daily intake. This technology is a foundation to produce folate-biofortified commercial varieties that can help to reduce hidden hunger, decreasing the number of cases of fetus malformations and other diseases.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"20"},"PeriodicalIF":1.9,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144015414","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}
Rong Yuan, Miao Wang, Zhen Li, Meiyan Hong, Li Su, Gang Wu, Xinhua Zeng
{"title":"A method for detecting transgenic rapeseed using pollen collected by Apis mellifera L.","authors":"Rong Yuan, Miao Wang, Zhen Li, Meiyan Hong, Li Su, Gang Wu, Xinhua Zeng","doi":"10.1007/s11248-025-00438-9","DOIUrl":"10.1007/s11248-025-00438-9","url":null,"abstract":"<p><p>With the continuous expansion of the planting area of genetically modified (GM) crops, the demand for efficient and comprehensive monitoring systems is becoming increasingly urgent. To establish a method suitable for large-scale monitoring of genetically modified rapeseed planting, beehives were strategically deployed at specific locations around genetically modified rapeseed fields, and the TaqMan quantitative PCR (qPCR) method was used to detect and analyze the genetically modified components in the rapeseed pollen collected by bees. The results demonstrated that the average Ct values for the CaMV35S promoter, Bar gene, NPTII gene, and HPT gene in the pollen of each hive were 27.91, 29.58, 31.49, and 31.97, respectively. The average ΔCt values for these four genes in hive pollen from 100 to 200 m were - 0.35, 1.66, 2.58, and 5.06, respectively, which were significantly lower than those from 300 to 1100 m (2.85, 4.01, 6.66, and 5.63). The results of this study have demonstrated the feasibility of using pollen collected by bees for large-scale detection of genetically modified rapeseed plants. This early warning model for GM crop spread based on bee pollination provides an efficient and practical solution for monitoring and managing genetically modified crops.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"18"},"PeriodicalIF":1.9,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789031","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}
Haiying Xiang, Guang Yuan, Chuhan Shi, Li Xu, Jianduo Zhang, Qili Mi, Qian Gao, Wenwu Yang, Haitao Huang, Kunmiao Wang, Wanli Zeng, Yang Ning, Qian Wang
{"title":"Shaker K<sup>+</sup> channel NKT3A enhances potassium uptake and transport in tobacco (Nicotiana tabacum L.) seedlings under low potassium stress.","authors":"Haiying Xiang, Guang Yuan, Chuhan Shi, Li Xu, Jianduo Zhang, Qili Mi, Qian Gao, Wenwu Yang, Haitao Huang, Kunmiao Wang, Wanli Zeng, Yang Ning, Qian Wang","doi":"10.1007/s11248-024-00419-4","DOIUrl":"10.1007/s11248-024-00419-4","url":null,"abstract":"<p><p>One of the nutrients that is necessary for plant growth and development is potassium (K<sup>+</sup>). The uneven production and distribution of global potassium resources significantly challenge crop yields and quality. A moderate increase in the potassium content within plants can enhance both crop yield and quality. This study identifies the Shaker K<sup>+</sup> channel NKT3A within the model crop, tobacco. The yeast heterologous expression system demonstrated its capability for K<sup>+</sup> inward transportation. GUS staining and RT-qPCR analyses of the constructed promoter materials revealed NKT3A's activity during the tobacco seedling stage. Expression levels are higher in the leaf and stems, with low potassium levels inducing upregulation of its expression, also observed in roots. Gene editing technology was employed to construct overexpression and knockout mutants, with subsequent measurement of their phenotypes. Results indicate that NKT3A expression enhances facilitates potassium absorption and transport in tobacco seedlings under low potassium conditions. For the first time, this article identifies the Shaker potassium channel gene NKT3A, which functions as an inward rectifier K<sup>+</sup> channel in tobacco. It elucidates the gene's role in regulating potassium distribution under low potassium conditions, thereby deepening our understanding of plant responses in such environments and offering a potential target for enhancing crop potassium use efficiency.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"17"},"PeriodicalIF":1.9,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143721573","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}
Yangjoo Kang, Deuk-Su Kim, Hyunjoo Hwang, Yerin Kim, Young-Jin Seo, Peter Hinterdorfer, Kisung Ko
{"title":"Plant-derived recombinant macromolecular PAP-IgG Fc as a novel prostate cancer vaccine candidate eliciting robust immune responses.","authors":"Yangjoo Kang, Deuk-Su Kim, Hyunjoo Hwang, Yerin Kim, Young-Jin Seo, Peter Hinterdorfer, Kisung Ko","doi":"10.1007/s11248-025-00433-0","DOIUrl":"10.1007/s11248-025-00433-0","url":null,"abstract":"<p><p>Prostatic acid phosphatase (PAP) is a specific protein that is highly expressed in prostate cancer. In this study, we constructed two recombinant PAP fusion genes: PAP fused to the immunoglobulin G (IgG) Fc fragment (designated PAP-Fc) and PAP-Fc fused to the endoplasmic reticulum retention sequence KDEL (designated PAP-FcK). Transgenic Nicotiana tabacum plants expressing these recombinant macromolecular proteins (MPs) were generated using Agrobacterium-mediated transformation, and the presence of both genes was confirmed through genomic PCR. Western blot analysis validated the expression of PAP-Fc and PAP-FcK MPs, which were successfully purified via protein A affinity chromatography. Size-exclusion high-performance liquid chromatography revealed dimeric peaks for PAP-Fc (PAP-Fc<sup>P</sup>) and PAP-FcK (PAP-FcK<sup>P</sup>). Bio-transmission electron microscopy demonstrated 'Y'-shaped protein particles resembling antibody structures. Moreover, PAP-Fc<sup>P</sup> and PAP-FcK<sup>P</sup> exhibited a high association rate with human FcγR and FcRn. Vaccination of mice with both PAP-Fc<sup>P</sup> and PAP-FcK<sup>P</sup> resulted in increased total IgG against PAP and enhanced activation of CD4<sup>+</sup> T cells, comparable to mice immunized with PAP, which served as a positive control. These findings indicate that both plant-derived MPs can effectively induce adaptive immunity, positioning them as promising candidates for prostate cancer vaccines. Overall, plants expressing PAP-Fc and PAP-FcK represent a viable production system for antigenic macromolecule-based prostate cancer vaccines.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"16"},"PeriodicalIF":1.9,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143721572","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":"Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).","authors":"Takashi Yamanaka, Akiko Sogo, Shingo Maegawa, Masato Kinoshita","doi":"10.1007/s11248-025-00434-z","DOIUrl":"10.1007/s11248-025-00434-z","url":null,"abstract":"<p><p>Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"15"},"PeriodicalIF":1.9,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711379","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}
Qiang Ding, Zhaokang Cui, Qianqian Shi, Yan Zhang, Nan He, Rihong Guo, Yu Tian, Shaoxian Cao, Jifeng Zhong, Huili Wang
{"title":"An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.","authors":"Qiang Ding, Zhaokang Cui, Qianqian Shi, Yan Zhang, Nan He, Rihong Guo, Yu Tian, Shaoxian Cao, Jifeng Zhong, Huili Wang","doi":"10.1007/s11248-025-00435-y","DOIUrl":"10.1007/s11248-025-00435-y","url":null,"abstract":"<p><p>β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLG<sup>c.61C > T</sup>), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLG<sup>c.61C > T</sup> mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLG<sup>c.61C > T</sup> single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"14"},"PeriodicalIF":1.9,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634684","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":"Genome editing research initiatives and regulatory landscape of genome edited crops in India.","authors":"Neha Sharma, Kanika Thakur, Rasna Zinta, Vikas Mangal, Dalamu, Jagesh Kumar Tiwari, Salej Sood, Som Dutt, Vinod Kumar, Brajesh Singh, Ajay Kumar Thakur","doi":"10.1007/s11248-025-00432-1","DOIUrl":"10.1007/s11248-025-00432-1","url":null,"abstract":"<p><p>Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"13"},"PeriodicalIF":1.9,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143626135","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}
Nasar Khan, Zhouyan Li, Akbar Ali, Biaohu Quan, Jindan Kang, Munib Ullah, Xi-Jun Yin, Muhammad Shafiq
{"title":"Comprehensive transcriptomic analysis of myostatin-knockout pigs: insights into muscle growth and lipid metabolism.","authors":"Nasar Khan, Zhouyan Li, Akbar Ali, Biaohu Quan, Jindan Kang, Munib Ullah, Xi-Jun Yin, Muhammad Shafiq","doi":"10.1007/s11248-025-00431-2","DOIUrl":"10.1007/s11248-025-00431-2","url":null,"abstract":"<p><p>Pigs are a vital source of protein worldwide, contributing approximately 43% of global meat production. Recent genetic advancements in the myostatin (MSTN) gene have facilitated the development of double-muscling traits in livestock. In this study, we investigate the transcriptomic profiles of second-generation MSTN-knockout (MSTN<sup>-/-</sup>) pigs, generated through CRISPR/Cas9 gene editing and somatic cell nuclear transfer (SCNT). Using RNA sequencing, we compared the transcriptomic landscapes of muscle tissues from MSTN<sup>-/-</sup> pigs and wild-type (WT) counterparts. The sequencing yielded an average unique read mapping rate of 86.7% to the Sus scrofa reference genome. Our analysis revealed 15,142 differentially expressed genes (DEGs), including 121 novel genes, with 2554 genes upregulated and 1629 downregulated in the MSTN<sup>-/-</sup> group relative to the wild-type group. Notable transcriptomic changes were identified in genes associated with muscle development, lipid metabolism, and other physiological processes. These findings provide valuable insights into the molecular consequences of MSTN inactivation, with potential applications in the optimization of livestock breeding and advancements in biomedical research.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"12"},"PeriodicalIF":1.9,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143469350","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}
Jie Sun, Yan Dong, Yuemei Meng, Jingran Bi, Hongmei Liu, Junjie Ren, Jinmao Wang, Yachao Ren, Minsheng Yang
{"title":"Effect of transgene on salt tolerance of tobacco.","authors":"Jie Sun, Yan Dong, Yuemei Meng, Jingran Bi, Hongmei Liu, Junjie Ren, Jinmao Wang, Yachao Ren, Minsheng Yang","doi":"10.1007/s11248-025-00430-3","DOIUrl":"10.1007/s11248-025-00430-3","url":null,"abstract":"<p><p>To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.</p>","PeriodicalId":23258,"journal":{"name":"Transgenic Research","volume":"34 1","pages":"11"},"PeriodicalIF":1.9,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053782","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}