Transgenic Research最新文献

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^{c.61C > T}), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLG^{c.61C > T} mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLG^{c.61C > T} 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.

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.

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^-/-) 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^-/- 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^-/- 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.

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.

The involvement of Loose Plant Architecture 1 (LPA1) in regulating plant growth and leaf angle has been previously demonstrated. However, the fundamental genetic background remains unidentified. To further understand the tissue expression profile of the NtLPA1 gene, an overexpression vector (pBI121-NtLPA1) was developed and employed to modify tobacco using the leaf disc method genetically. Validation confirmed the generation of transgenic tobacco plants with NtLPA1 overexpression. The findings indicated that increased NtLPA1 overexpression substantially decreased plant auxin sensitivity and modulated signal transduction and polar transport, significantly reducing leaf angle, diminished leaf area during early and late growth stages, and shortened root length. In summary, NtLPA1 augmented tobacco resistance to severe shin disease by modulating the expression of disease-associated genes PBZ1, PR1b, and the growth regulator auxin polar transport factor PIN1.

Drought, as an abiotic stressor, globally limits cereal productivity, leading to early aging of leaves and lower yields. The expression of the isopentenyl transferase (IPT) gene, which is involved in cytokinin (CK) biosynthesis, can delay drought-induced leaf senescence. In this study, the Agrobacterium Isopentenyl transferase (IPT) gene was introduced into two local hexaploid wheat cultivars, NR-421 and FSD-2008. The expression cassette was developed containing the IPT gene under transcriptional regulation of the stress-inducible promoter 'Dehydrin,' sourced from Hordeum vulgare. The gene expression cassette was assembled in pSB219M, a modified transformation vector for monocots, equipped with both an antibiotic (spectinomycin) and an herbicide selection marker (BASTA). Initial screening of transgenic plants involved BASTA selection (2 and 3 mg/L) and was subsequently confirmed through PCR analysis. The transformation efficiencies of NR-421 and FSD-2008 were 0.4% and 0.3%, respectively. The qRT-PCR analysis under stress conditions showed a 13.5-fold higher expression of the IPT gene in T₂ transgenic plants of NR-421 and a 5.8-fold higher expression in those of FSD-2008 than in non-transgenic controls. Under stress conditions, the wheat transgenic plants exhibited increased chlorophyll and relative water content. Additionally, for total soluble proteins, two transgenic lines from the NR-421 variety showed a significant increase, whereas no notable change was observed in the FSD-2008 transgenics. Moreover, the transgenic lines displayed increased plant height, higher fresh and dry biomass, and increased seed weight compared to the non-transgenic controls. These findings highlight that stress-inducible expression of the IPT gene in wheat leads to enhanced grain yield and subsequently improved drought tolerance.

Lignin is a crucial defense phytochemical against phytophagous insects. Cinnamoyl-CoA reductase (CCR) is a key enzyme in lignin biosynthesis. In this study, transgenic Populus davidiana × P. bolleana overexpressing the PdbCCR gene were generated via Agrobacterium-mediated transformation. Successful integration of PdbCCR into the poplar genome was confirmed by PCR amplification and quantitative reverse transcription PCR (qRT-PCR). The lignin content in the transgenic poplar leaves was significantly higher than that in the wild poplar, and after L. dispar larvae fed on the transgenic poplar, the CCR activity was clearly induced. The L. dispar larvae grew slowly after feeding on transgenic poplar and the laccase, cellulase and three detoxifying enzymes were induced compared with larvae after feeding on wild-type poplar. The bioassay further revealed that transgenic poplar plants overexpressing PdbCCR showed a high level of resistance to L. dispar larvae. These results confirmed that PdbCCR is a candidate gene for breeding insect resistant poplar.

Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRAS^G12D that enables the "activation" of KRAS^G12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated Kras^RR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the Kras^RR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRAS^G12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRAS^G12D function is effectively suppressed by specific inhibitor MRTX1133.

The transcription factor Tbx20 is integral to heart development and plays a significant role in various cardiac diseases. Despite its established importance, the regulatory mechanisms and functional significance of Tbx20 remain incompletely understood. To elucidate these mechanisms, we initially conducted eQTL mapping to identify genetic loci associated with Tbx20 expression in heart tissue from BXD mice. Co-expression and enrichment analyses revealed pathways linked to Tbx20, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and FoxO signaling. Additionally, protein-protein interaction studies identified essential cardiac proteins, such as Myl2 and Myl7, along with upstream regulators like Mef2c. To validate our bioinformatic findings, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess the relative mRNA expression levels of TBX20 and Mef2c in the heart tissues of BXD mice compared to their parental strains (B6 and D2). Our results demonstrated significant up-regulation of both TBX20 and Mef2c in the BXD group relative to the parental strains. Conversely, both genes were down-regulated in B6, D2, Control, and Treatment groups when compared to BXD mice. These findings confirm the predicted regulatory roles of TBX20 and Mef2c in cardiac development as suggested by our initial analyses.This study not only reinforces the critical role of Tbx20 in cardiac gene regulation but also highlights its potential as a therapeutic target for cardiovascular disorders. Further investigations into Tbx20 and its interactions will enhance our understanding of heart biology and contribute to the development of targeted therapies for heart diseases.

The environmental risk assessment (ERA) of genetically modified (GM) crops in Japan requires collecting data from a comparative study of a GM and non-GM control in an in-country confined field trial (CFT). This in-country CFT requirement is used to address concerns that differences in the local environmental conditions may lead to differences in growth and/or risks of GM crops. However, this requirement for in-country CFT has recently been exempted for certain GM maize and GM cotton traits, and instead CFT data from other countries are used to inform the ERA of these GM events. However, in-country CFTs continue to be required for GM B. napus. Our objective is to assess whether using B. napus as a host crop increases the potential for differences between GM B. napus and conventional B. napus that may have an impact on biodiversity occurring only under the Japanese environment. In this paper agronomic data was compiled from seven local CFTs of GM B. napus events to assess the potential for differences between GM and non-GM B. napus for three key areas; competitiveness, potential to produce harmful substances, and outcrossing. Considering these elements, the need for conducting CFTs locally for ERA of future GM B. napus traits is discussed. The assessment concluded that conducting CFT locally is not necessary for GM B. napus events if traits do not bring competitive advantage or produce harmful substances only under Japanese environment.