Objective:Heat-induced stress (HS) amid global warming compromise cattle reproductive performance causing economic loss including the vulnerable Korean native beef cattle, Hanwoo. Nevertheless, knowledge of response and effect in cattle oviduct epithelial cells (OEC) under HS remains lacking due to restricted commercial cell line and multi-omics data available. This research attempts to develop immortalized OEC derived from Hanwoo cattle as HS cell culture model and to investigate its HS-specific response by utilizing biomarkers comprising transcriptomic and proteomic analysis. Methods:Primary OEC was isolated from fresh Hanwoo oviduct and immortalized by using piggyBac transposon-mediated SV40T expression system. HS optimization was performed by detection of reactive oxygen species (ROS) and oxidative-endoplasmic reticulum (ER) stress biomarkers under varied HS and recovery time (4, 12, and 24 h). Optimized period was applied to generate differentially expressed genes (DEG) and protein (DEP) analyzed using gene ontology (GO) and pathway enrichment. Results:Immortalized OEC was successfully developed with high CDH1 positivity (98.2%) sorting. Increased intracellular ROS was detected at 4 h HS followed by rapid substantial upregulation of HSP70 and BiP protein and delayed upregulation of oxidative stress genes (SOD1, CAT, GPX1) peaked at 24 h HS period. In total, 510 DEG (fold change ≥|2|) and 258 DEP (fold change ≥|1.5|) were significantly altered under 24 h HS condition. Furthermore, GO and pathway enrichment analysis of the DEG-DEP list revealed upregulation processes related to oxidative-thermal stress and protein folding. Other altered processes involve immune response, cellular senescence, response to starvation, and extracellular matrix structures as enriched terms suggesting possible hindering effects to OEC reproductive role. Conclusion:The immortalized OEC provides a stable system to evaluate HS response in cattle oviducts. Transcriptomic and proteomic data from this study offer valuable resources for targeted molecular approach to attenuate detrimental HS effect in cattle reproduction.

Macrophages are primary regulators of the innate immune system, integrating pattern recognition receptor (PRR) signaling and cytokine production to shape early inflammatory processes. However, the molecular mechanisms governing macrophage-driven responses in chickens remain poorly understood, despite their importance in controlling major infectious diseases. Among these pathways, interleukin (IL)-1β functions as a central mediator of inflammatory signaling, yet its integration with Toll-like receptor (TLR) pathways in chicken macrophages remains unclear. Here, lipoteichoic acid (LTA)-Toll-like receptor (TLR)2, poly(I:C)-TLR3, and lipopolysaccharide (LPS)-TLR4 pathways were investigated in the chicken macrophage-like cell line HD11 and an IL1β-deficient HD11 mutant, with PBMC-derived macrophages included for comparison. HD11 cells exhibited minimal responsiveness to LTA–TLR2 stimulation, whereas LPS–TLR4 stimulation elicited a limited response primarily characterized by strong induction of IL-1β and IL-8 but minimal or undetectable IL-6, tumor necrosis factor (TNF)-α, IL-18, and interferon (IFN)-β. In contrast, poly(I:C)–TLR3 stimulation induced IL-1β, IL-18, and IFN-β with limited induction of other cytokines. A genetically engineered CRISPR/Cas9 IL1B knockout (KO) HD11 model has been established to evaluate molecular interactions of the inflammatory response, which appears to be modulated by IL-1β in a TLR ligand-specific context. The present study highlights distinct pattern recognition profiles on chicken macrophages that may offer a molecular basis for IL-1β-targeted signaling networks and immunomodulation in chickens.

OBJECTIVE:Heat stress (HS) negatively affects cattle reproduction, decreasing livestock productivity. HS response in cattle reproductive cumulus cells remain understudied due to limited cell lines and comprehensive omics data. This study aims to establish immortalized cumulus cells from Hanwoo cattle as a HS model and explores their HS response using molecular markers including transcriptomics and proteomics approaches. METHODS:Immortalized cumulus cells were established from primary cumulus isolated from fresh Hanwoo cattle follicles by using piggyBac transposon-mediated SV40T expression system. HS condition was optimized using reactive oxygen species (ROS) detection, quantitative reverse-transcription polymerase chain reaction, and Western blotting on oxidative and endoplasmic reticulum stress markers under different HS and recovery periods (4 h, 12 h, and 24 h). Transcriptomic and proteomic data were generated to identify HS-related differentially expressed genes (DEG) and proteins (DEP) utilized for gene ontology (GO) and pathway enrichment analysis. RESULTS:We established the first immortalized cumulus cell line with high specificity (99% CD44[+]) for in vitro HS study. ROS accumulation was observed at 4 h HS, leading to immediate upregulation of HSP70 and BiP proteins, with oxidative stress gene expression (SOD1, CAT, GPX1) peaked at 12 h HS. We identified 754 DEGs (fold change≥|2|) and 357 DEPs (fold change≥|1.5|) related to HS in the cumulus cell line. Enrichment analysis exhibited upregulation in GO and pathways related to protein unfolding and cellular response to HS, respectively, from both the DEG and DEP list. Downregulated DEG and DEP showed significant enrichment in GO related to the extracellular matrix, potentially affecting cumulus-oocyte complex communication for reproductive function. CONCLUSION:The cumulus cell line serves as a robust in vitro model for studying HS response in cattle reproductive system. The multi-omics data could help clarify the comprehensive HS response and identify possible molecular targets to mitigate negative HS effects in the future.

In brief:The CD40 ligand-CD40 signaling system expressed at the maternal-conceptus interface in pigs is involved in regulating maternal immunity by modifying endometrial endothelial cell function during early pregnancy. This paper reveals the role of CD40 ligand-CD40 signaling at the maternal-conceptus interface in pigs. Abstract:To successfully establish and maintain pregnancy in pigs, a variety of factors must work together at the maternal-conceptus interface to form an immune environment appropriate for both the mother and the conceptus. Our transcriptomics study has shown that cluster of differentiation ligand 40 (CD40L) and its receptor CD40, which are known to play important roles in regulating cell- and antibody-mediated immunity, are expressed in the endometrium during early pregnancy. However, the roles of the CD40L and CD40 signaling system are not well understood. Therefore, we determined the expression, regulation and function of CD40L and CD40 at the maternal-conceptus interface in pigs. The endometrium expressed CD40L and CD40 mRNAs at the greatest levels on day 15 of pregnancy. The CD40L protein was localized predominantly to luminal epithelial cells on day 15 of pregnancy, and the CD40 protein was found in luminal epithelial, stromal and vascular endothelial cells in the endometrium during pregnancy. During early pregnancy, the conceptus expressed CD40 but not CD40L and chorioallantoic tissues during mid- to late pregnancy expressed both CD40L and CD40. Interferon-γ increased the expression of CD40L and CD40 in endometrial explants. CD40L increased the migration but not the proliferation of cultured porcine uterine endothelial (pENDO) cells in vitro. In addition, CD40L affected the expression of genes related to angiogenesis, cell adhesion, chemokines and immunity in pENDO cells. These results suggest that the CD40L-CD40 system might play an important role in the establishment of pregnancy in pigs by regulating endometrial endothelial cell function during the implantation period.

Programmed cell death (PD) ligand 1 (PDL1) and PDL2 and their receptor PD1 play critical roles in immunity, and an involvement of the PDL1/PDL2-PD1 signaling system in the immune tolerance during pregnancy has been suggested in humans. However, the role of the PDL1/PDL2-PD1 signaling system at the maternal-conceptus interface has not been studied in pigs. Thus, this study determined the expression, regulation, and function of the PDL1/PDL2-PD1 signaling pathway at the maternal-conceptus interface in pigs. The endometrium expressed PDL1, PDL2, and PD1 mRNAs in a pregnancy stage-specific manner with greater levels of expression on Day 15 of pregnancy than on Day 15 of the estrous cycle. PDL1 protein was localized to some stromal cells and vascular smooth muscle cells, PDL2 protein to luminal (LE) and glandular epithelial (GE) and endothelial cells, and PD1 protein to LE, GE, and some stromal cells, but not in T lymphocytes and endothelial cells. The conceptus tissues during early pregnancy and chorioallantoic tissues during mid- to late pregnancy expressed PD1, PDL1, and PDL2. Interferon-γ (IFNG) increased the expression of PDL1 and PDL2, but not PD1, in endometrial explants. PDL1 and PDL2 increased the expression of inhibitors of apoptosis in porcine uterine epithelial cells overexpressing PD1. These results suggest that the expression of PDL1 and PDL2 are induced by IFNG of conceptus origin and PDL1/PDL2-PD1 signaling may play an important role in maintaining the epithelial stability by regulation of apoptosis and controlling the endometrial environment for the establishment and maintenance of pregnancy in pigs. SUMMARY: The PDL1/PDL2-PD1 signaling system induced by conceptus-derived IFNG plays an important role in maintaining endometrial epithelial stability by increasing the epithelial expression of inhibitors of apoptosis during early pregnancy.

In a previous study, we identified a hypermuscular phenotype attributed to both muscle fiber hyperplasia and hypertrophy in myostatin (MSTN)-knockout (KO) chickens generated by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) technology. In mammals, MSTN deletion results in increased production of fast glycolytic fibers, accompanied by decreased production of slow oxidative fibers. However, the effects of MSTN deletion on muscle fiber types have rarely been studied in avian species such as chickens. In this study, we analyzed muscle fibers in chickens at various ages to investigate whether MSTN deletion alters muscle fiber composition after hatching. Immunofluorescence and histochemical staining, including ATPase and nicotinamide adenine dinucleotide hydrogen-tetrazolium reductase (NADH-TR) staining, were performed on biceps femoris (BF) tissues of wild-type (WT) and MSTN-KO chickens at hatching and at 10 and 18 weeks. BF muscle fibers were classified into three types (1, 2A, and 2B). MSTN-KO chickens produced more muscle fibers than did WT chickens at all ages. Fiber hypertrophy was not observed in MSTN-KO chickens at hatching, but was detected in all fiber types at 10 and 18 weeks. Compared to WT chickens, there was a significant decrease in Type 1 fibers and an increase in Type 2B fibers in the BF tissues of MSTN-KO chickens, whereas the amount of Type 2A fibers remained unchanged. Furthermore, MSTN deletion led to upregulated expression of genes in the glycolytic pathway and MyoD in skeletal muscles. Thus, MSTN deletion altered muscle fiber composition in chickens, inducing Type 2B fiber dominance and hyperplasia during the embryonic stage, whereas muscle hypertrophy due to nutritional uptake became predominant after hatching.

Heat stress is a major environmental challenge that compromises reproductive performance in cattle, particularly under the intensifying conditions of global climate change. This review provides a comprehensive overview of how heat stress impairs bovine reproduction at the physiological, cellular, and molecular levels and explores practical strategies for mitigation. In females, heat stress disrupts hormonal regulation, estrous behavior, and ovulation, and diminishes oocyte and embryo quality, resulting in reduced conception rates and increased pregnancy losses. In males, prolonged heat exposure impairs spermatogenesis and semen quality and delays post-stress recovery. Dairy cattle are especially vulnerable due to the elevated metabolic demands of lactation. Although beef cattle exhibit greater thermotolerance, they also experience reduced fertility under prolonged heat stress, particularly during breeding and early gestation. At the cellular level, heat stress triggers oxidative damage, mitochondrial dysfunction, immune dysregulation, and altered epigenetic and cytoskeletal dynamics. Integrative transcriptomic analyses across key reproductive tissues reveal both conserved and cell type-specific molecular responses. These include activation of inflammatory and apoptotic pathways, suppression of chaperone-mediated protein folding and hormone receptor signaling, and downregulation of uterine receptivity programs. To counter these effects, current strategies involve environmental modifications, genetic selection for thermotolerance, and supportive treatments such as antioxidants, methyl donors, and hormonal protocols. However, most interventions remain symptomatic and nonspecific. Future efforts must prioritize mechanistically grounded approaches that target the molecular drivers of heat-induced reproductive dysfunction. Continued research integrating multi-omics, network-based modeling, and pharmacological discovery will be critical to developing next-generation solutions that enhance reproductive resilience and sustainability in cattle production systems.

Objective: The study aimed to improve the efficiency of leghorn male hepatoma (LMH) cells for animal virus vaccine production by transitioning from adherent to suspension culture and evaluating the effects of dextran sulfate (DS) on preventing cell aggregation. The goal was to enhance cell growth, viability, and glucose metabolism and to develop efficient suspension-adapted LMH cells for large-scale vaccine production.Methods: LMH cells previously cultured in an adherent state were transferred to 125 mL Erlenmeyer flasks to conduct suspension culture. Cell culture performance, including cell density, viability, and glucose metabolism, during the cultures was measured, along with an assessment of cell aggregation. Additionally, mRNA expression levels of genes associated with cell adhesion and apoptosis were monitored.Results: DS supplementation in suspension culture enhanced cell viability and growth, with higher cell densities and viabilities compared to control media. Additionally, DS supplementation reduced glucose consumption and waste production, indicating improved metabolic efficiency. DS also delayed cell aggregation, possibly by downregulating integrin expression and promoting anti-apoptotic gene expression. However, even after 2 months, cell aggregation persisted in both control and DS-supplemented cultures, suggesting further optimization is needed for LMH cell adaptation to suspension culture.Conclusion: DS supplementation in LMH cell suspension cultures led to notable improvements in cell growth, viability, and glucose metabolism, while also decreasing the cell aggregation.

OBJECTIVE:Germ cell identity is regulated by the coordinated action of multiple key transcription factors during embryonic development, which includes the induction and control of germ-line-specific gene expression. The expression of DEAD-box helicase 4 (DDX4) and deleted in azoospermia-like (DAZL) genes in chickens plays a pivotal role in germplasm formation and the specification of germ cell lineage from a totipotent genome. This study aimed to investigate the regulatory mechanisms underlying germ cell fate determination. METHODS:Large-scale gene expression profiling was conducted to screen and select critical transcription factors. This analysis identified differentially expressed genes in chicken primordial germ cells (PGCs), comprising 1,020 transcription factors. Additionally, we generated a chicken DF1 cell line featuring an enhanced green fluorescent protein (eGFP) reporter precisely knocked into the transcriptional start site of the DAZL gene using the CRISPR-Cas9 system, enabling real-time monitoring of DAZL expression during reprogramming. RESULTS:Through analysis of transcription factor binding sites within approximately 10 kb upstream regions of DDX4 and DAZL, resulting in the selection of 10 candidate transcription factors for germ cell induction. Subsequently, the ten transcription factors identified as regulators of germ cell identity were transduced into the DAZL-knock-in eGFP DF1 cells. This approach led to the successful induction of eGFP-expressing cells in vitro, driven by the endogenous DAZL promoter. We conducted further characterization of these cells to confirm their germ cell-specific properties. CONCLUSION:Our findings offer new insights into the transcriptional regulation of chicken germ cells by identifying key factors that activate DAZL expression. These results indicated valuable opportunities for advancing germ cell induction from somatic cells, with potential applications of in vitro models for studying germ cell-specific gene regulatory pathways in avian species.

ABSTRACT Background Muscle diseases are serious challenges to human health. Prokineticin receptor 1 (PROKR1) has emerged as a potential target to improve muscle function through increasing oxidative muscle fibres, but there are no clinically applicable synthetic PROKR1 agonists. Methods Drugs with biological properties of prokineticin 2 (PK2) were discovered through connectivity map (CMap) analysis. Their effects on PROKR1 were evaluated using molecular docking, PROKR1 signalling and competitive binding assays. Pregnant dams were fed diets containing varying celecoxib concentrations (0, 500, 1000 and 1500 ppm) from gestation day 5 through weaning. Offspring were given high‐fat diets (HFD) from weaning until 20 weeks old, and body composition, insulin resistance, energy expenditure, exercise performance and histological analysis of muscle tissues were evaluated. Results Celecoxib, with a connectivity score of 64.19 to PK2 and a docking score of −9.0 to PROKR1, selectively activated Gs signalling at 4 μM of EC50 and increased NR4A2 protein levels by 1.6‐fold (p < 0.01) in PROKR1‐overexpressing cells. It competitively inhibited PK2 binding to PROKR1 and reduced cAMP accumulation. In murine and human myotubes, celecoxib increased Prokr1 protein levels by 1.8‐fold (p < 0.05), pCreb by 1.5‐fold (p < 0.05) and Nr4a2 by 1.3‐fold (p < 0.05). It also elevated Myh7 index by 2.2‐fold (p < 0.0001), mitochondrial content by 1.6‐fold (p < 0.001) and fatty acid oxidation (FAO) activity by 4.1‐fold (p < 0.05). Offspring exposed to celecoxib during pre‐ and postnatal muscle development exhibited activated Prokr1 signalling, enhanced oxidative muscle fibre formation and improved muscle phenotype despite HFD. At weaning, both male and female offspring showed dose‐dependent increases in lean mass (> 9.35%, p < 0.001) and grip strength (< 18.0%, p < 0.01). At 12 weeks old, mice displayed a dose‐dependent decrease in weight loss (> 13.3%, p < 0.05), increased lean mass (> 16.2%, p < 0.05), improved insulin resistance (> 70.4%, p < 0.0001), energy expenditure (> 173%, p < 0.0001) and grip strength (> 23.5%, p < 0.001). Celecoxib also increased Myh7‐positive muscle fibre composition (> 10.8%, p < 0.05) and mitochondrial mass (> 32.8%, p < 0.05) in the gastrocnemius and soleus muscles, accompanied by significant Prokr1 signalling activation. These effects persisted in both male and female mice at 20 weeks old. Conclusions Celecoxib shows promise as a PROKR1 agonist and clinically applicable exercise mimetic for the treatment of muscular disorders.

Retinoic acid inducible gene I (RIG-I) is an innate immune RNA sensor which can detect viral infection such as influenza viruses. Duck but not chicken has an RIG-I gene. However, the immune responses could be induced in chicken cells by transferring the duck RIG-I transgene. However, effects of other pathogen-recognition receptor (PRR) genes such as Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5) could not be ruled out. In this study, we knocked out TLR3 and MDA5 genes using gene-editing protocol, and stably transferred the duck RIG-I transgene into TLR3/MDA5 double knockout (KO) chicken DF1 cells. We investigated the antiviral responses induced by duck RIG-I in chicken cells. Duck RIG-I induced the expression of interferon-stimulated genes (ISGs) and inflammatory cytokines such as interferon regulatory factor 7 (IRF7), interferon β (IFNβ), Mx1, and protein kinase R1 (PKR1) after treatment with polyinosinic: polycytidylic acid (poly I:C) in TLR3/MDA5 double KO DF1 cells. Additionally, to examine the duck RIG-I signaling cascade, we knocked out mitochondrial antiviral-signaling protein (MAVS), which encodes an antiviral signaling factor in innate immunity. Duck RIG-I in TLR3/MDA5/MAVS triple KO DF1 cells did not activate downstream expression of ISGs. Finally, to analyze the global signaling pathways of duck RIG-I in chicken cells, next-generation sequencing of total mRNAs with and without poly I:C treatment was conducted. In conclusion, duck RIG-I mediated antiviral signaling independently of TLR3 and MDA5, and MAVS induced and stimulated ISGs by duck RIG-I in chicken cells.

Despite growing concerns about the adverse effects of antibiotics in farm animals, there has been little investigation of the effects of florfenicol in laying hens. This study examined the effect of florfenicol on the intestinal homeostasis, immune system, and pathogen susceptibility of laying hens. The oral administration of florfenicol at field-relevant levels for 5 d resulted in a decrease in the gut microbiota genera Lactobacillus, Bacillus, and Bacteroides, indicating the development of intestinal dysbiosis. The dysbiosis led to decreased mRNA levels of key regulators peroxisome proliferator-activated receptor gamma (PPAR-γ) and hypoxia-inducible factor-1α (HIF-1α), compromising intestinal hypoxia. Intestinal homeostasis was also disrupted, with decreased expression of Occludin and Mucin 2 (Muc2) genes combined with increased gut epithelial permeability. The breakdown in intestinal homeostasis and immune function provided a favorable environment for opportunistic bacteria like avian pathogenic Escherichia coli (APEC), culminating in systemic infection. Immunologically, florfenicol treatment resulted in increased proportion and absolute number of MRC1L-B+ monocytes/macrophages in the spleen, indicating an exacerbated infection. Furthermore, both the proportion and absolute number of γδ T cells in the lamina propria of the cecum decreased. Treatment with florfenicol reduced butyrate levels in the cecum. However, the administration of butyrate before and during florfenicol treatment restored factors associated with intestinal homeostasis, including PPAR-γ, Occludin, and Muc2, while partially restoring HIF-1α, normalized intestinal hypoxia and gut permeability, and reversed immune cell changes, suppressing APEC systemic infection. The uncontrolled and widespread use of florfenicol can negatively affect intestinal health in chickens. Specifically, florfenicol was found to impair intestinal homeostasis and immune function in laying hens, including by reducing butyrate levels, thereby increasing their susceptibility to systemic APEC infection. The development of strategies for mitigating the adverse effects of florfenicol on gut health and pathogen susceptibility in laying hens is therefore essential.

Background Acute kidney injury (AKI) has a complex pathophysiology and imposes serious health concerns worldwide. Extracellular vesicles (EVs) derived from induced mesenchymal stem cells (iMSCs) have been recognized as novel cell-free therapeutics for various inflammatory and degenerative disorders. In this study, we investigated whether iMSCs stimulated with a pan-peroxisome proliferator-activated receptor (PPAR) agonist could enhance the therapeutic efficacy of EVs against AKI. Methods Human iMSCs were primed with or without lanifibranor, a PPAR agonist for 24 h, and EVs were collected after an additional 24 h. The basic characteristics of EVs were evaluated using cryo-transmission electron microscopy imaging, immunoblot detection of EV markers, nanoparticle tracking analysis, and localization in AKI kidneys. In vitro, the potential of the EVs to promote the growth and survival of HK-2 cells undergoing cisplatin-induced apoptosis and anti-inflammatory effects in M1-polarized THP-1 was compared. Subsequently, AKI was induced in BALB/c mice using cisplatin. After 8 and 24 h of cisplatin treatment, iMSC-EVs or pan-PPAR-iMSC-EVs were injected intravascularly. At 96 h after cisplatin administration, the renoprotective effects of iMSC-EVs or pan-PPAR-iMSC-EVs in inhibiting inflammation and apoptosis were compared using serum biochemistry, histology, immunohistochemistry, and gene expression analysis by qPCR. Results Both EV types expressed EV markers and had typical EV morphology, and their localization in the renal tissue was confirmed. The proliferation and survival of HK-2 cells were higher in pan-PPAR-iMSC-EVs than those in iMSC-EVs. In M1-polarized THP-1 cells, the reduction in the mRNA expression of inflammatory cytokines was more significant in pan-PPAR-iMSC-EVs than that in iMSC-EVs. In the mouse model of cisplatin-induced AKI, pan-PPAR-iMSC-EVs markedly enhanced renoprotective effects compared to iMSC-EVs. Specifically, pan-PPAR-iMSC-EVs reduced tissue inflammation, immune cell infiltration, and apoptosis. Pan-PPAR-iMSC-EVs also increased renal capillary density. Conclusion Priming iMSCs with a PPAR agonist significantly improved the therapeutic potential of EVs by reducing inflammation and apoptosis. The reported strategy may contribute to the development of a novel cell-free option for AKI treatment. Trial registration: Not applicable.

Abstract The integration of big data analytics with cancer research is catalyzing a transformative approach in cancer treatment, primarily focusing on the discovery of novel and efficacious anticancer targets. Our study presents an advanced algorithm, specifically crafted to exploit the extensive data available in the field of oncology. We started with the genomic and clinical information of 8,864 patients with 33 different cancers (TCGA). Then we implemented the following algorithm to discover anti-cancer targets by analyzing the clinical significance (cBioPortal), drug development status (Cortellis), and oncogenicity (DepMap) of candidate genes: Candidate genes = {gene | gene ∈ Genes, [Frequency(gene) > 50, Drug(gene) ∈ {'biological testing', 'preclinical stage'}, Association(gene) ≥ 0.4, Publication(gene) ≤ 200] ∨ [Publication(gene) ≥ 200 ∧ Boolean(gene)]}. We employed this algorithm to analyze fusion genes, which represent promising anti-cancer targets known for their potential to elicit substantial clinical responses, but there is a high demand for new ones. We identified four druggable therapeutic targets out of a total of 15,291 fusion genes through the algorithm: frame-shifted FGFR3-TACC3, in-framed DLK1-RPS11, frame-shifted CHP1-RAD51B, and in-framed TBC1D22A-SMYD3. We conducted in vitro validation studies of these fusion genes in NIH3T3 cell lines, and it confirmed that all of the fusion genes not only produce mRNA and protein levels but also induce oncogenic effects on cellular behavior. In the case of FGFR3-TACC3, the introduced fusion gene induced mRNA (p < 0.05) and protein expression (p < 0.05) even when frame-shifted. In addition, the proliferation rate of transformed cells increased more than 4-fold on day 10 (p < 0.0001) and colony formation increased more than 5-fold on day 21 (p < 0.01) compared to wild-type cells. These results demonstrate the tumorigenicity of the fusion genes. Taken together, this study emphasizes the crucial role of big data in propelling oncology research forward. The algorithm we developed can offer a new pathway for creating innovative cancer treatment, marking a significant advancement in the realm of personalized cancer therapy. Citation Format: Dooho Kim, Jong Woo Park, Jung-Ae Kim, Jeong-Hoon Kim, Tae Sub Park, Joonghoon Park. Big data-driven discovery of novel oncogenic fusion genes for anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5954.

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

Innate immunity, as an organism's first line of defense, plays a crucial role in rapidly responding to and protecting the body against invading pathogens. As a cytosolic RNA sensor for viral infections, including infections caused by influenza virus, the innate immune system in chickens has 2 major pathogen-recognition receptors (PRRs): Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5). The signaling pathways activated by PRRs are complex, systemic processes that underlie the response to foreign molecules. In this study, we investigated the interactions among MDA5, mitochondrial antiviral signaling protein (MAVS), and stimulator of interferon genes (STING) signaling in chicken cells. To exclude the effects of TLR3, we transfected the clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) expression vector and TLR3-targeted gRNA plasmid into chicken DF-1 cells. We selected TLR3-knockout (KO) cell line and sequentially, we established 2 double-KO cell lines: TLR3-MAVS KO and TLR3-STING KO. After treatment with polyinosinic:polycytidylic acid (poly(I:C)), type I interferon (IFN), IFN-stimulated gene, and antiviral gene (IFN regulatory factor 7, IFNβ, Mx1, and protein kinase R1) expression was not completely activated in TLR3-MAVS KO cells, whereas it was consistently upregulated in wild-type and TLR3-STING KO DF-1 cells. These results suggest that STING is not an intermediator between MDA5 and MAVS; moreover, it does not directly interact with MDA5 during innate immune activation in chicken DF-1 cells.

Objective The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. Methods By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. Results We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of ZGFP-knockin chickens. By mating ZGFP-knockin females (ZGFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (ZGFP/Z) even before hatching. Conclusion Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.