Transgenic Lines Research Articles

Background: One of the core pathological features of myocardial infarction is the loss of cardiomyocytes, and promoting cardiomyocyte regeneration represents a potential therapeutic strategy for post-infarction heart failure.The aim of this research is to uncover the role and mechanisms of Receptor-like Protein LRTM1 in cardiomyocyte regeneration, providing novel therapeutic targets for cardiac repair. Method: In this study, we performed two cardiac injury models - apical resection and myocardial infarction operation - in both neonatal and adult mice. Cardiomyocyte-specific Lrtm1 deletion was achieved through combinatorial use of Myh6-MerCreMer and Myh6-iCre transgenic mouse lines with Lrtm1 fl/fl conditional alleles. A series of molecular signaling experiments, including RNA sequencing, immunostaining and coimmunoprecipitation , were conducted. Heart regeneration and cardiac function were evaluated by Masson staining and echocardiography, respectively. Result: Integrated analysis of RNA-seq data from P1 and P7 murine hearts, as well as P1 myocardial infarction (MI) versus sham-operated hearts, revealed a negative correlation between LRTM1 expression and cardiomyocyte proliferative capacity. Notably, LRTM1 exhibited cardiac-specific enrichment, particularly in cardiomyocytes. These findings implicate LRTM1 as a critical regulator of intrinsic myocardial regeneration. In vitro and in vivo functional studies demonstrated that LRTM1 knockdown enhanced proliferation in neonatal rat primary cardiomyocytes, extended the postnatal cardiomyocyte proliferative temporal window in neonatal mice, and promoted cardiomyocyte proliferation in adult MI mice, thereby improving post-MI cardiac function and attenuating pathological ventricular remodeling. Mechanistically, combined RNA-seq and motif enrichment analyses revealed that LRTM1 downregulation suppressed JAK/STAT1 signaling activation by impairing extracellular IFN-α/receptor binding, leading to reduced IRF1 transcription factor expression. This cascade ultimately drives cardiomyocyte proliferation through coordinated modulation of cell cycle-related proteins. Conclusion: Cardiomyocyte-specific knockout of LRTM1 promotes cardiomyocyte proliferation, extends the proliferative time window in neonatal mice and ameliorates post-infarction cardiac remodeling in adult mice via interferon signaling.These findings support a potentially important new therapeutic approach for human heart failure

Introduction: Designer receptors exclusively activated by designer drugs (DREADDs) are a robust chemogenetic tool for the interrogation of cellular electrical activity. Particularly, hM4Di activates the Gi signaling pathway, and can induce cellular hyperpolarization, leading to decreased electrical activity. Our aim is to engineer human induced pluripotent stem cell-derived cardiomyocytes (hiSPC-CMs) with controllable electromechanical properties to elucidate the role of transplanted hiPSC-CMs in cardiac regeneration. We hypothesize that activating hM4Di in transgenic hiSPC-CMs will inhibit their electrical activity. Methods: We developed a new hiPSC line expressing DREADD using the PiggyBac system (PB). The PB transposon included the hM4Di gene under the cTnT promoter, with mCherry and puromycin resistance genes as transfection markers ( Fig 1A ). After establishing the line, we differentiated these hiPSCs into DREADD-expressing cardiomyocytes (hiPSC-CM M4Di ). We activated hM4Di using clozapine N-oxide (CNO) and determined the optimal concentration through a concentration swipe. Optical mapping was used to analyze the electrical activity of hiPSC-CM M4Di . Results: After transfection, we observed the expression of mCherry in hiPSCs ( Fig 1B ). Additionally, we confirmed that these cells retained their pluripotency by evaluating the stemness markers Sox2 and Nanog ( Fig 1C ). Then, we performed directed cardiac differentiation. The cells showed spontaneous beating after day 8 of differentiation. We determined that the optimal concentration for hiPSC-CM M4Di inhibition was 200µM CNO, based on concentration swipe. Furthermore, our optical mapping data showed that spontaneous beating rate was decreased 30 minutes after adding CNO and completely stopped at 60 minutes. The spontaneous beating recovered after CNO was removed. CNO did not affect the electrical behavior of wild type cells (Fig 1D) . Conclusions: We developed a transgenic hiPSC line expressing DREADD. The produced hiPSC-CM M4Di responded positively to the CNO ligand, allowing inhibition of their electromechanical activity. This cell line holds potential for investigating the electrophysiology of grafted hiPSC-CMs, and for application as disease model in vitro.

The adaptability of plants to drought involves tolerance and recovery. Alfalfa (Medicago sativa L.), a key forage crop, is highly susceptible to water deficit. This study found that key pathways, including starch and sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis, were activated under drought and rewatering. Key module analysis identified 4 hub genes (CIPK2, MYB6, bZIP43, and NF-YB3), and revealed that the expression of CIPK2 continuously increased during drought stress. Functional validation in Arabidopsis confirmed that MsCIPK2 enhanced drought resistance by promoting root growth, lateral root development, and stomatal closure. Physiologically, transgenic lines exhibited increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, maximum quantum efficiency of PSII (Fv/Fm), photosynthetic efficiency (ΦPSII), photochemical quenching (qP), and nonphotochemical quenching (NPQ) and reduced malondialdehyde (MDA) and relative electrolyte conductivity (REC). Mechanistically, MsCIPK2 mediated these physiological alterations by modulating the expression of key genes such as APX1, PP2C, ARF7, LEA14 and others. These findings provide molecular insights into alfalfa drought adaptation and a biotechnological basis for improving its resistance.

Background/Objectives: Bacterial blight (BB) represents one of the most devastating diseases threatening global rice production. Exploring and characterizing disease resistance (R) genes provides an effective strategy for controlling BB and enhancing rice resilience. Common wild rice (Oryza rufipogon) serves as a valuable reservoir of genetic diversity and disease resistance resources. In this study, we identified and functionally characterized a novel NLR gene, YPR1, from common wild rice (Oryza rufipogon), which exhibited significant spatial, temporal, and tissue-specific expression patterns. Methods: Using a combination of conventional PCR, RT-PCR, bioinformatics, transgenic analysis, and CRISPR/Cas9 gene-editing approaches, the full-length YPR1 sequence was successfully cloned. Results: The gene spans 4689 bp with a coding sequence (CDS) of 2979 bp, encoding a 992-amino acid protein. Protein domain prediction revealed that YPR1 is a typical CNL-type NLR protein, comprising RX-CC_like, NB-ARC, and LRR domains. The predicted molecular weight of the protein is 112.43 kDa, and the theoretical isoelectric point (pI) is 8.36. The absence of both signal peptide and transmembrane domains suggests that YPR1 functions intracellularly. Furthermore, the presence of multiple phosphorylation sites across diverse residues implies a potential role for post-translational regulation in its signal transduction function. Sequence alignment showed that YPR1 shared 94.02% similarity with Os09g34160 and up to 96.47% identity with its closest homolog in the NCBI database, confirming that YPR1 is a previously unreported gene. To verify its role in disease resistance, an overexpression vector (Ubi–YPR1) was constructed and introduced into the BB-susceptible rice cultivar JG30 via Agrobacterium tumefaciens-mediated transformation. T1 transgenic lines were subsequently inoculated with 15 highly virulent Xanthomonas oryzae pv. oryzae (Xoo) strains. The transgenic plants exhibited strong resistance to eight strains (YM1, YM187, C1, C5, C6, T7147, PB, and HZhj19), demonstrating a broad-spectrum resistance pattern. Conversely, CRISPR/Cas9-mediated knockout of YPR1 in common wild rice resulted in increased susceptibility to most Xoo strains. Although the resistance of knockout lines to strains C7 and YM187 was comparable to that of the wild type (YPWT), the majority of knockout plants exhibited more severe symptoms and significantly lower YPR1 expression levels compared with YPWT. Conclusions: Collectively, these findings demonstrate that YPR1 plays a crucial role in bacterial blight resistance in common wild rice. As a novel CNL-type NLR gene conferring specific resistance to multiple Xoo strains, YPR1 provides a promising genetic resource for the molecular breeding of BB-resistant rice varieties.

Transcriptome analysis of OsNCED3 transgenic rice reveals the response mechanism to alkaline stress.

Over-expression of CsHAK4 from tea plant (Camellia sinensis L.) negatively regulates high salt tolerance in transgenic Arabidopsis.

TaHAK1 promotes salt tolerance via synergistic modulation of K+/Na+ ion homeostasis and auxin signaling in rice.

Disruption of mitochondrial homeostasis and apoptosis by oryzalin exposure in zebrafish embryos.

Functional characterization of tonoplast sugar transporter gene HpTST1 involved in soluble sugars accumulation during fruit development process of red pitaya (Hylocereus polyrhizus).

In CA1 hippocampus, pyramidal cells (PCs) can be classified as deep or superficial based on their radial position within the stratum pyramidale. Deep and superficial PCs form biased circuits with perisomatic-targeting PV+ basket cells, but it is unknown if such cell-type-specific circuit motifs extend to dendrite-targeting interneurons. Using male and female mice, we investigated synaptic connectivity and physiology in brain slices from four transgenic lines thought to capture distinct subsets of interneurons: SST-IRES-Cre, Nkx2.1-Cre, Chrna2-Cre, and Htr3a-GFP. First, we found that oriens-lacunosum moleculare (OLM) cells captured by the Chrna2-Cre line are a subset of Htr3a-GFP+ cells in the hippocampus. This novel finding is consistent with previous work showing Nkx2.1-Cre OLM cells are distinct from both Chrna2-Cre and Htr3a-GFP+ OLM cells. Indeed, in paired whole-cell recordings, Nkx2.1-Cre+ interneurons in the stratum oriens, but not Chrna2-Cre+ or Htr3a-GFP+ cells, received more excitatory synaptic connections from superficial PCs relative to deep PCs. Next, we expressed channelrhodopsin in interneurons to investigate inhibition along the proximal and distal dendrites of PCs. We found that superficial PCs received stronger inhibition along their proximal dendrites than deep PCs from SST+ interneurons. Furthermore, this circuit motif was dependent on layer but not PC projection class. Finally, Chrna2-Cre OLM cells provided stronger inhibition to the distal dendrites of deep PCs relative to superficial PCs. Our data reveal that superficial and deep PCs engage in cell-type-specific circuits with dendrite-targeting interneurons. Furthermore, they support that Nkx2.1-Cre OLM cells and Chrna2-Cre/Htr3a-GFP OLM cells are distinct subtypes that form unique circuits in CA1.Significance statement Region CA1 is the primary output circuit from the hippocampus to the rest of the brain during learning and memory recall. CA1 contains subtypes of excitatory pyramidal cells that are distinguished by their radial position as deep or superficial, and several subtypes of inhibitory interneurons that target specific regions of pyramidal cells to control their output and synaptic integration. Determining if these cells form unique circuits is necessary to understand the logic of how information flows through CA1 to other brain regions. Here, we mapped synaptic connections and their physiology between specific types of pyramidal cells and interneurons that control dendritic inhibition. We provide a new circuit diagram of cell-type-dependent connections and their strengths in CA1.

Lignin constitutes a fundamental component of plant defense mechanisms against environmental stressors. 4-coumarate 3-hydroxylase (C3H) serves as a pivotal enzyme in lignin biosynthesis. However, its role in the halophyte Sesuvium portulacastrum remains uncharacterized. In this study, the SpC3H gene was cloned, and subsequent sequence alignment and phylogenetic analyses revealed the highest similarity (57.14%) with BvC3H from Beta vulgaris, exhibiting the closest evolutionary relationship with Beta vulgaris and Spinacia oleracea C3H protein. Quantitative real-time polymerase chain reaction demonstrated that SpC3H expression was markedly upregulated in both roots and leaves of S. portulacastrum under 800 mM NaCl treatment. Root expression peaked at 48 h (25.3-fold), whereas leaves displayed dual expression maxima at 12 h (7.9-fold) and 72 h (10.7-fold). Subcellular localization assays confirmed cytoplasmic distribution. Heterologous expression in Arabidopsis thaliana indicated that transgenic lines exhibited enhanced growth performance, higher fresh weight, and elevated lignin contents relative to wild-type plants under salt stress, accompanied by reduced reactive oxygen species (ROS) accumulation and lower relative electrical conductivity. Furthermore, activities of superoxide dismutase and peroxidase, together with expression of lignin biosynthesis-associated and antioxidant enzyme genes, were markedly elevated. Collectively, these findings establish that SpC3H confers salt tolerance by promoting lignin biosynthesis and activating antioxidant defenses to eliminate ROS, thereby providing a theoretical foundation for genetic improvement of plant salt tolerance.

ABSTRACTAimHeart failure is a clinical syndrome where the heart's structural or functional impairment leads to inadequate blood flow to meet the body's metabolic demands. Mitochondrial dysfunction is increasingly recognized as a central contributor underlying the contractile impairment observed in the failing heart. This study aimed to explore the interplay between calcium dynamics, cardiac mechanical performance, and mitochondrial ATP production during the progression of heart failure in zebrafish larvae exposed to chronic isoproterenol stimulation.MethodsHeart failure was induced by treating zebrafish larvae with 100 μM isoproterenol from 3 to 14 days postfertilization (dpf). Cardiac calcium transients, contractility, and mitochondrial ATP levels were assessed in vivo using transgenic lines expressing specific fluorescent biosensors. Additionally, transcriptomic analysis by RNA sequencing was performed on hearts collected at 14 dpf following prolonged isoproterenol exposure.ResultsAfter 4 days of isoproterenol treatment (7 dpf), larvae exhibited ventricular dilation, reduced calcium levels, and diminished contractile force (p < 0.0001), although cardiac output remained intact. In contrast, extended treatment (11 days; 14 dpf) led to decompensated heart failure, characterized by a significant decline in cardiac output (p < 0.0001). Mitochondrial ATP levels were preserved at 7 dpf but dropped markedly at 14 dpf (p < 0.0001). Transcriptomic profiling at this later stage revealed downregulation of key functions (p < 0.05) involved in mitochondrial energy metabolism and energy transfer.ConclusionIn this model, heart dysfunction was initially evidenced by cardiac dilation. At 4 days of isoproterenol treatment, calcium levels and contractility decreased. Subsequently, decompensation coincided with a collapse in mitochondrial ATP production.

BackgroundDevelopment ornamental varieties with enhanced floral traits and distinctive characteristics is a central goal in floriculture. Anthocyanins, the principal flavonoid pigments in floral tissues, are synthesized via the well-characterized flavonoid biosynthesis pathway. Chalcone isomerase (CHI) catalyzes the first committed reaction in the flavonoid biosynthetic pathway.MethodIn this study, we investigated the effects of CHI suppression via RNA interference (RNAi) in three Petunia hybrida cultivars exhibiting distinct petal colors. Leaf discs from three Petunia hybrida lines exhibiting distinct petal colors (blue, pink, and purple) were inoculated with Agrobacterium tumefaciens harboring the recombinant plasmid pBI121 containing the RNAi construct targeting Phchi. Transgenic plants were regenerated on MS medium via microshoots emerging from wounded explant regions and verified by PCR using two primer pairs specific to the CHI silencing vector. Gene expression analysis was conducted for chi and associated pigment biosynthesis genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and dihydroflavonol 4-reductase (DFR), comparing transgenic and wild-type plants. Chalcone and naringenin contents were quantified using a NanoDrop ELISA plate reader. Data analysis was performed using SPSS version 16, and mean comparisons were evaluated via T-test at a significance level of α = 0.05.ResultsTransformation efficiencies for CHI-silenced Petunia lines were 57.89%, 72.0% and 84.0% for the pink, blue, and purple phenotypes, respectively. Phenotypic evaluation revealed not only altered pigment distributions but also novel floral morphologies, such as tetramerous corollas and twisted tubular petal margins, in the CHI-suppressed lines. Quantitative metabolic profiling demonstrated a significant reduction in naringenin levels across all transgenic lines, whereas, chalcone accumulation was significantly elevated in a subset of lines. The expression of CHI gene was markedly reduced in all lines carrying the CHI-RNAi construct; however, transcript levels did not differ significantly between the aberrant floral phenotypes and the control. Transcript levels of key downstream flavonoid biosynthesis pathway genes, including F3H, F3'H, F3'5'H, and DFR were significantly attenuated in CHI-silenced lines compared with wild-type controls. This concerted down‐regulation indicates that suppression of the CHI‐catalyzed step exerts a broader repressive effect on subsequent pathway components. Despite the overall repression of flavonoid-biosynthetic genes in CHI-silenced lines, certain novel phenotypes maintained or even increased expression of specific downstream enzymes. Notably, blue-flowered transformants exhibited F3'H transcript levels comparable to wild-type control, and pink-flowered lines retained F3'5'H expression. These exceptions highlight the nuanced, tissue-specific regulatory responses triggered by CHI suppression.ConclusionCollectively, our results demonstrate that RNAi- mediated silencing of CHI gene is an effective strategy for generating novel pigmentation patterns and floral morphology in Petunia hybrida. Moreover, this work provides critical insights into how targeted manipulation of a single enzymatic step can influence the broader transcriptional network governing anthocyanin biosynthesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07607-2.

The effector AGLIP1 hijacks peroxidase OsAPX8 to coordinate rice immune suppression and ROS-Driven cell death for facilitating Rhizoctonia solani infection.

ABSTRACTThe ability to manipulate neurons that express corticotropin‐releasing factor (CRF) or its receptors has led to many new discoveries about how CRF signaling modulates stress‐related behaviors in mice. However, similar advances in rats have been limited by the lack of reporter lines targeting CRF receptors, restricting access to behavioral paradigms that are more suitable for, or specific to, rats. Here, we provide an extensive neuroanatomical characterization of a recently generated CRFR1‐Cre rat. We show that Cre and tdTomato are expressed in a pattern that closely aligns with previously reported CRFR1 expression patterns, as determined by in situ hybridization in rats and by genetic labeling in CRFR1 transgenic mice. We detail expression patterns at multiple rostrocaudal levels to validate the use of this transgenic rat in multiple neural circuits that signal via CRF activation of CRFR1. Furthermore, we demonstrate that expression of Cre recombinase, combined with viral vector delivery, can be used to trace CRFR1‐expressing neurons and their axonal terminals in target regions. This study is intended to serve as a resource for examining the expression pattern of CRFR1 and as a validation of the CRFR1‐Cre rat for reliable use with Cre‐dependent viral approaches to manipulate and/or record the activity of CRFR1‐expressing neurons in distinct brain regions. We expect that the availability of the CRFR1‐Cre rat, along with an increasing number of transgenic rat lines, will further expand our understanding of how CRF transmission mediates stress responses by leveraging the behavioral strengths of the rat as a model system.

Seed vigor is an important trait ecologically, agronomically, and economically, and is controlled by manifold genetic and exogenous factors. Dehydration-Responsive Element-Binding Protein 2B (DREB2B), a subgroup of the DREB transcription factor family, is well-known for conferring multiple abiotic stress resistance. However, the role of DREB2B in seed vigor has not been identified. Here, DREB2B was identified as a negative regulator of seed vigor using a loss-of-function mutant, gene editing, and over-expressing transgenic lines studies in Arabidopsis and Gossypium spp.. The lower and higher sensitivity of loss-of-function mutants and overexpression lines of DREB2B to Abscisic Acid (ABA) and Fluridone, respectively, emphasized the negative roles of DREB2B in seed vigor and germination via the ABA-mediated pathway. Further genetic and molecular analyses revealed that DREB2B exhibits both synergistic and independent functions in regulating seed germination and vigor concerning ABA INSENSITIVE 3 (ABI3). We observed that DREB2B formed transcriptional complexes with RADICAL-INDUCED CELL DEATH1 (RCD1) and Similar to RCD One 1 (SRO1) to regulate seed germination and vigor. In addition, RNA-seq analysis of dreb2b and rcd1-3 lines indicated that DREB2B and RCD1 may target the same pathways in seed germination and vigor associated with ABA accumulation modification, which is supported by DREB2B directly regulating ABA DEFICIENT 2 (ABA2) promoter activity. Collectively, these results suggest that ABA-mediated complexes consisting of DREB2B, RCD1, SRO1, and ABI3 function upstream of ABA2 to negatively regulate seed vigor in plants, expanding on our knowledge of seed development.

Loss of function variants of SCN1B are associated with a range of developmental and epileptic encephalopathies (DEEs), including Dravet syndrome. These DEEs feature a wide range of severe neurological disabilities, including changes to social, motor, mood, sleep, and cognitive function which are notoriously difficult to treat, and high rates of early mortality. While the symptomology of SCN1B-associated DEEs indicates broad changes in neural function, most research has focused on epilepsy-related brain structures and function. Mechanistic studies of SCN1B/Scn1b have delineated diverse roles in development and adult maintenance of neural function, via cell adhesion, ion channel regulation, and other intra- and extra-cellular actions. However, use of mouse models is limited as knockout of Scn1b, globally and even in some cell-specific models (e.g., Parvalbumin+ interneuron-specific knockout) in adult mice, leads to severe and progressive epilepsy, health deterioration, and 100% mortality within weeks. Here, we report findings using male and female mice of a novel transgenic line in which Scn1b was specifically deleted in cerebellar Purkinje cells. Unlike most existing models, these mice survive and thrive. However, we quantified marked decrements to Purkinje cell physiology as well as motor, social, and cognitive dysfunction. Our data indicates that cerebellar Purkinje cells are an important node for dysfunction and neural disabilities in SCN1B-related DEEs, and combined with previous work identify this as a potentially vital site for understanding mechanisms of DEEs and developing therapies that can treat these disorders holistically.Significance Statement Developmental and epileptic encephalopathies (DEEs), caused SCN1B or other gene variants, cause neuropsychiatric disabilities including movement, social, and cognitive dysfunction. Because global Scn1b knockout mice die in the third week of life and many DEE disabilities localize to cerebellar function we crossed a conditional Scn1b knockout mouse model with a cerebellar Purkinje cell-specific Cre line to study post-developmental neurological function. We found that Purkinje cell-specific Scn1b knockout mice survived into adulthood but had severe loss of Purkinje cell excitability, ataxia, decreased social interest, and disrupted cognitive performance. Our study shows that Scn1b is vital to the function of cerebellar neurons and loss of this gene isolated to Purkinje cells is sufficient to cause multiple disabilities that mirror SCN1B-associated DEEs.

Identification of YABBY transcription factors in foxtail millet and functional characterization of SiFILs under GA and drought conditions.

Ionizing radiation (IR) induced sex-specific reproductive and offspring developmental toxicity in Zebrafish.

In plants, responses to hypoxia include activation of fermentation pathways, cytosolic acidification, and other metabolic shifts. In Arabidopsis (Arabidopsis thaliana), the transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) contributes to regulating cellular responses to low-oxygen stress; however, the underlying mechanism remains largely unknown. Here, we showed that transgenic lines overexpressing STOP1 exhibited improved tolerance of hypoxia and submergence, whereas knockout of STOP1 reduced tolerance. STOP1 accumulated during hypoxia and was degraded during post-hypoxia reoxygenation via ubiquitination by PLANT U-BOX-TYPE UBIQUITIN LIGASE 24 (PUB24). Under hypoxia, MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 interacted with and phosphorylated STOP1 to compete with its PUB24-mediated ubiquitination, thus stabilizing STOP1 in the nucleus, where it activated the transcription of GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2 for cellular homeostasis of acidic metabolism during hypoxia. Mutating three phosphorylated residues in STOP1 to alanine attenuated its nuclear accumulation and diminished STOP1-mediated hypoxia tolerance. Moreover, we identified the lipid phosphatidic acid as a critical modulator of the MPK3/6-STOP1 association. Overall, these findings uncover an antagonistic biochemical mechanism in which MPK3/6-dependent phosphorylation and PUB24-dependent ubiquitination of STOP1 modulate its nuclear accumulation to control hypoxia responses in Arabidopsis.