Biochemical Genetics最新文献

{"title":"Association of miR-146a/b and miR-181a with Chronic Obstructive Pulmonary Disease.","authors":"Sanaz Abbaspour-Aghdam, Amir Valizadeh, Khadijeh Pouya, Sajad Jafarzadeh, Haleh Mikaeili, Mehdi Nadiri, Khalil Hajiasgharzadeh, Majid Ahmadi, Armin Sadeghi","doi":"10.1007/s10528-026-11405-2","DOIUrl":"10.1007/s10528-026-11405-2","url":null,"abstract":"<p><p>miRNAs play critical roles in regulating inflammatory responses in chronic obstructive pulmonary disease (COPD). This study investigated the expression patterns of miR-146a/b and miR-181a, along with associated inflammatory mediators, in COPD patients. This cross-sectional study included 40 patients with acute exacerbation of COPD (AECOPD), 40 patients with stable COPD, and 40 healthy controls. Expression levels of miR-146a, miR-146b, and miR-181a in peripheral blood mononuclear cells (PBMCs) were quantified using quantitative real-time PCR. Serum concentrations of high-mobility group box 1 (HMGB1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured by ELISA. Cytokine mRNA expression was assessed in PMA-stimulated PBMCs. Multivariable logistic regression was performed to adjust for potential confounders including age and smoking pack-years. Expression of miR-146a, miR-146b, and miR-181a was significantly decreased in both COPD and AECOPD groups compared to controls (all p < 0.001), with further reduction in AECOPD patients. Multivariable analysis demonstrated that decreased miR-146a (OR = 2.45, 95% CI: 1.58-3.81, p < 0.001) and miR-181a (OR = 2.18, 95% CI: 1.42-3.35, p < 0.001) remained independent predictors of COPD after adjusting for age and smoking exposure. Serum HMGB1, IL-6, and TNF-α levels were significantly elevated in COPD patients, while IL-10 showed decreased expression. PMA-stimulated PBMCs from COPD patients demonstrated enhanced pro-inflammatory cytokine production capacity. In exploratory ROC analysis, miR-146a showed moderate discriminatory capacity (AUC = 0.78, 95% CI: 0.65-0.91). Dysregulation of miR-146a/b and miR-181a is independently associated with COPD, even after accounting for smoking exposure. These findings suggest potential roles in disease pathophysiology, though prospective validation in larger, independent cohorts is required before clinical applicability can be determined.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7482-7500"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148154680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fisetin Modulates Chemoresistance-Associated miR-21, miR-181a, miR-203, miR-101 and miR-381 in Capecitabine-Resistant HT29 Colorectal Cancer Cells.","authors":"Zehra Kanlı, İrem Peker Eyüboğlu, Kazim Yalcin Arga, Banu Aydın, Can Erzik, Hülya Cabadak","doi":"10.1007/s10528-026-11412-3","DOIUrl":"10.1007/s10528-026-11412-3","url":null,"abstract":"<p><p>Capecitabine is a key component of colorectal cancer (CRC) chemotherapy; however, acquired resistance markedly limits its efficacy. MicroRNAs (miRNAs) are central regulators of drug response, and the flavonoid fisetin has been reported to exert anti-tumor and chemosensitizing effects. Here, we investigated whether fisetin modulates the expression of chemoresistance-associated miRNAs in a capecitabine-resistant CRC cell model. Candidate miRNAs were prioritized from the publicly available dataset GSE30894 using differential expression analysis, which identified miR-21, miR-181a, miR-203, miR-101, and miR-381 as differentially expressed between sensitive and resistant colorectal cancer cells. These miRNAs were quantified by RT-qPCR in parental HT29 and capecitabine-resistant HT29 (CR/HT29) cells under basal conditions and after 24-h exposure to capecitabine (40 µM), fisetin (120 µM), or the combination. Nuclear morphology was assessed by DAPI staining. At baseline, CR/HT29 cells showed lower expression of miR-381, miR-21, miR-203, and miR-101 and a trend towards higher miR-181a compared with parental HT29 cells. Treatment with capecitabine, fisetin, or their combination differentially modulated these miRNAs in the two cell lines. In both parental and resistant cells, fisetin-containing regimens reduced miR-21, while in CR/HT29 cells, fisetin significantly decreased miR-181a expression compared with fisetin-treated parental cells. Fisetin and fisetin + capecitabine also altered miR-203 and miR-101 levels, with a more pronounced suppression in HT29 than in CR/HT29 cells. These changes were accompanied by marked morphological alterations in resistant cells. Fisetin modulates chemoresistance-associated miRNAs in colorectal cancer, particularly in capecitabine-resistant HT29 cells, suggesting a chemosensitizing adjuvant role and offering an in vitro basis for in vivo studies.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7695-7719"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148222269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MiR-146b-5p/ERBB4 Axis Drives LPS-induced Acute Kidney Injury by Modulating NF-κB/p65.","authors":"Dazhuang Sun, Limin Wang, Shoulei Liu","doi":"10.1007/s10528-026-11386-2","DOIUrl":"10.1007/s10528-026-11386-2","url":null,"abstract":"<p><p>We aimed to explore the role of miR-146b-5p in lipopolysaccharide (LPS)-induced acute kidney injury. C57BL/6N mice were intraperitoneally injected with LPS to establish an acute kidney injury model, and the effects of miR-146b-5p inhibition on AKI progression in vivo were explored. The renal function of mice was evaluated by determining serum creatinine and blood urea nitrogen levels. Kidney histopathology was examined using H&E and PAS staining. Cell apoptosis in vivo was analyzed via TUNEL staining, and the TUNEL-positive cells per field were quantified. HK-2 cells were stimulated with LPS (1 µg/mL) to replicate an inflammatory AKI environment in vitro. To investigate function and molecular mechanisms of miR-146b-5p, HK-2 cells were transfected with mimics and inhibitor of miR-146b-5p. Apoptosis in HK-2 cells was assessed via flow cytometry using Annexin-V/FITC and propidium iodide (PI) staining, while levels of inflammatory cytokine (IL-1β, IL-6, TNF-α) were measured using ELISA. RNA pull-down and luciferase assays were used to explore interaction between miR-146b-5p and ERBB4 and activation of the downstream NF-κB/p65 signaling was investigated via western blot. The results showed that miR-146b-5p expression was upregulated by ~ 133% in the LPS-induced AKI mouse model (p < 0.01) and increased up to ~ 137% in LPS-treated HK-2 cells (p < 0.001) compared to control. Inhibition of miR-146b-5p improved renal function, and BUN levels were reduced by ~ 48% (p < 0.01) and creatinine levels were decreased by ~ 40% compared to LPS + NC antagomir group (p < 0.01). Inhibition of miR-146b-5p also attenuated tissue injury, suppressed cell apoptosis, with the TUNEL-positive cells reduced by 46% compared to LPS + NC antagomir group (p < 0.01), and lowered inflammatory cytokine levels, with IL-1β reduced by ~ 54% (p < 0.01), IL-6 reduced by ~ 55% (p < 0.01), and TNF-α reduced by 51% (p < 0.01) compared to LPS + NC antagomir group. Mechanistically, miR-146b-5p directly targeted ERBB4, whose suppression reversed the protective effects of miR-146b-5p inhibition. Importantly, we found that the miR-146b-5p/ERBB4 axis regulates the NF-κB/p65 signaling pathway, providing a potential mechanistic link between miRNA activity and inflammation in AKI. In conclusion, this study reveals that the miR-146b-5p/ERBB4 axis promotes the activation of the NF-κB pathway and drives apoptosis and inflammation in LPS-induced AKI, suggesting miR-146b-5p as a novel therapeutic target for AKI.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7410-7433"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148040549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Promoter Methylation of HOXA5 and NDRG2 in Multiple Myeloma Patients with Renal Impairment.","authors":"Riham Abdel-Hamid Haroun, Dina Samir Elsaid, Mona A Al-Dosoky, Tamer A Elbedewy, Sally M Gamal-Eldin, Rabab A Keshk","doi":"10.1007/s10528-026-11401-6","DOIUrl":"10.1007/s10528-026-11401-6","url":null,"abstract":"<p><p>Renal impairment (RI) is frequently caused by multiple myeloma (MM), which makes clinical care more difficult if renal function deteriorates or renal failure develops. Thus, in this investigation, we looked at the relationship between HOXA5&NDRG2 promoter methylation in MM patients and evaluated their prognostic potential in predicting RI risk in MM patients. This study included 60 control volunteers and 120 MM patients. The promoter methylations of HOXA5&NDRG2 were quantitatively evaluated by real-time PCR (qPCR) after the extraction of gDNA from whole blood and then treated with bisulfite. The promoter methylation percentages of HOXA5&NDRG2 were significantly increased in MM patients (77.16 ± 2.43&45.49 ± 2.16;p < 0.05) when compared to controls (4.47 ± 0.61&3.33 ± 0.31,respectively), in stage II&III patients when compared to stage I patients and in patients with age ≥ 60 years when compared to patients with age < 60 years. While only the methylation percentage of NDRG2 promoter was significantly increased in MM patients with RI (53.61 ± 3.21,p < 0.05) when compared to MM patients without RI (37.37 ± 2.04). Results obtained from ROC curve revealed that both HOXA5&NDRG2 promoter methylation were good diagnostic tools for MM. The NDRG2 promoter methylation was good prognostic biomarker could predict renal while HOXA5 promoter in the prediction of MM staging. The Kaplan-Meier survival test showed that patients with higher HOXA5&NDRG2 promoter methylation had a shorter OS and a worse prognosis. Only NDRG2 promoter hypermethylation was significantly associated with the risk of RI development in MM patients. HOXA5&NDRG2 promoter hypermethylation may have roles in the molecular etiology of MM and could be used as a treatment regimen.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7542-7558"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148196825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Epirubicin-Induced Upregulation of miR-143-3p and miR-145 Enhances Cytotoxicity in MCF-7, MDA-MB-231, and BT-474 Breast Cancer Cell Lines.","authors":"Morteza Rajabi, Niki Ghambari Mohammadi, Mahsa Mombeyni, Roudabeh Behzadi Andouhjerdi","doi":"10.1007/s10528-026-11408-z","DOIUrl":"10.1007/s10528-026-11408-z","url":null,"abstract":"<p><p>Drug resistance poses a significant challenge to effective breast cancer chemotherapy. This study investigated the regulatory effects of Epirubicin on miR-143-3p and miR-145 expression in BT-474, MCF-7, and MDA-MB-231 breast cancer cell lines. These miRNAs, known as tumor suppressors, are often downregulated in breast carcinoma, correlating with poorer prognosis. Cells were treated with Epirubicin, and miR-143-3p and miR-145 expression was quantified via qRT-PCR. Epirubicin IC₅₀ values were 0.42 µg/mL (MCF-7), 2.94 µg/mL (MDA-MB-231), and 0.86 µg/mL (BT-474) after 48 h, demonstrating dose- and time-dependent cytotoxicity. Treatment significantly upregulated miR-143-3p and miR-145 expression, with MCF7 cells showing a 3.5-fold increase in miR-145 (p < 0.01) and a 2.8-fold increase in miR-143-3p (p < 0.05) compared to controls. Cell viability decreased by up to 70% at 6 µg/mL Epirubicin in MCF-7 cells, with apoptosis rates rising to 8.65% (MCF-7) and 9.75% (MDA-MB-231) after 24 h (p < 0.01). Colony formation was reduced by 60-80% across cell lines (p < 0.001), and wound closure was inhibited by 40-50% in MDA-MB-231 cells at 24 h (p < 0.0001). These findings suggest that Epirubicin enhances therapeutic efficacy by upregulating miR-143-3p and miR-145, potentially mitigating drug resistance. This highlights their potential as therapeutic targets for improving outcomes in resistant breast cancer subtypes.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7587-7607"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148196849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HILPDA, Regulated by WTAP in an m6A-Dependent Manner, Enhances Osteosarcoma Development by Inhibiting Ferroptosis and Promoting Glycolysis.","authors":"Yong Jing, Guoyong Zhang, Chenghui Sun, Yawei Pan, Jun Tuo, Zhi Li, Chengji Jiang, Zejian Qiu, Qin Lou","doi":"10.1007/s10528-026-11404-3","DOIUrl":"10.1007/s10528-026-11404-3","url":null,"abstract":"<p><p>Osteosarcoma (OS) is the most common primary malignant bone cancer. Hypoxia-inducible lipid droplet-associated protein (HILPDA) promotes tumor growth, migration, and ferroptosis resistance in OS. However, the oncogenic mechanisms of HILPDA in OS are largely unexplored. Glycolytic activity was assessed by measuring glucose uptake and lactate release. Ferroptosis was evaluated by measuring ROS, MDA, Fe²⁺, and total iron contents. Cell proliferation was assessed via EdU incorporation assay, and tube formation ability was analyzed. Animal experiments were used to test the function in vivo. Expression analysis was performed using quantitative PCR, immunoblotting, and immunohistochemistry. The regulation of Wilms' tumor 1-associating protein (WTAP) in HILPDA was detected by methylated RNA immunoprecipitation (MeRIP) assay. HILPDA and WTAP were upregulated in OS tissues. Overexpression of HILPDA or WTAP was associated with poor prognosis in OS patients. HILPDA deficiency impaired OS cell proliferation and glycolysis while promoting ferroptosis, concurrently attenuating HUVEC tube formation in vitro. Moreover, HILPDA depletion constrained the in vivo growth of HOS OS xenografts. Mechanistically, WTAP regulated HILPDA expression through an m6A-dependent manner. Additionally, HILPDA re-expression reversed sh-WTAP-mediated alterations in OS cell glycolysis and ferroptosis and HUVEC tube formation. Our findings identify the WTAP/HILPDA axis as a key regulator of OS development through coordinated control of glycolysis, ferroptosis, and angiogenesis, offering new avenues for multi-targeted therapeutic intervention in OS.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7678-7694"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148215709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Analysis of Stemness and Differentiation Gene Expression in Mesenchymal Stem Cells from Permanent and Deciduous Teeth: An In Vitro Study to Evaluate Implications for Regenerative Dentistry.","authors":"Seyed Amir Abas Noorbakhsh, Fatemeh Pourmohammadi-Nejad, Ali Pirsadeghi, Aliakbar Yousefi-Ahmadipour","doi":"10.1007/s10528-026-11411-4","DOIUrl":"10.1007/s10528-026-11411-4","url":null,"abstract":"<p><p>Dental pulp is a valuable source of mesenchymal stem cells (MSCs), offering significant promise for cell therapy and regenerative medicine, particularly in treating oral and dental conditions. For clinical translation, understanding which dental stem cell source provides superior proliferative and differentiation capacity for specific tissue regeneration (bone vs. dentin) is essential. This study aimed to compare the gene expression profiles and osteogenic potential of MSCs derived from permanent (DPSCs) and deciduous (SHED) teeth. DPSCs and SHED were isolated and characterized using flow cytometry. Their differentiation potential into bone and fat cells was assessed, along with colony formation, doubling time (DT), cell viability, alkaline phosphatase (ALP) activity, and the expression of stemness genes (SOX2, OCT4, NANOG) and differentiation genes (OCN, DSPP, AMBN, SPARC, IBSP). SHEDs showed higher colony formation (33.66 vs. 28.33) and shorter doubling time (33.13 h vs. 39.25 h) compared to DPSCs. Importantly, SHEDs exhibited significantly higher ALP activity after osteogenic induction, confirming their superior early osteogenic differentiation potential. SHEDs also expressed stemness genes (OCT4, NANOG, SOX2) and osteogenic markers (AMBN, OCN) at higher levels, while DPSCs showed higher expression of odontogenic markers (DSPP, SPARC, IBSP). Overall, SHEDs in this study showed greater proliferative capacity, faster doubling times, and enhanced early osteogenic potential compared to DPSCs, whereas DPSCs showed stronger odontogenic differentiation potential. These findings suggest that SHEDs may be more suitable for bone and enamel-like tissue regeneration, while DPSCs are better suited for dentin-pulp complex repair, providing a rationale for stem cell selection in regenerative dentistry.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7720-7742"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148222199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Universal, Cost-Effective, and Efficient rRNA Depletion Method for Bacterial mRNA Enrichment.","authors":"Ravi Chauhan, Hardi Patel, Seema Rawat","doi":"10.1007/s10528-026-11362-w","DOIUrl":"10.1007/s10528-026-11362-w","url":null,"abstract":"","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7743-7758"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147607796","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Role of Small Segmental Duplications in Generating Identical Isoforms Through Alternative Splicing Sites.","authors":"Ulkar Huseynova Mustafayeva, Sevda Babayeva, Orkhan Mustafayev, Latifa Hasanli, Irina Goldenkova-Pavlova, Afat Mammadova","doi":"10.1007/s10528-026-11394-2","DOIUrl":"10.1007/s10528-026-11394-2","url":null,"abstract":"<p><p>Alternative splicing plays a crucial role in expanding proteomic diversity but can also generate identical isoforms under certain conditions. While mutually exclusive splicing of tandem exons has occasionally been reported to produce identical isoforms, the extent to which other splicing events contribute to this phenomenon remains unclear. In this study, we demonstrate that alternative 5' and 3' splice site selection can also lead to the formation of identical isoforms, providing an additional type of splicing event for functional redundancy in transcriptomes. To address this, we analyzed reference genome annotations from 15 plant species, including Arabidopsis thaliana and wheat (Triticum aestivum), obtained from the RefSeq database. Identical isoforms were computationally defined as transcripts with distinct exon-intron structures but identical coding sequences. Our analysis reveals that the majority of alternative 5' and 3' fragments originate from small segmental duplications, suggesting that sequence repetition within gene regions facilitates the emergence of such splicing patterns. We also observed differences in the annotated 5' UTRs of some identical isoforms. However, since the alternative splicing sites themselves were not located within UTRs, these differences may reflect annotation uncertainty rather than genuine AS-derived variation. Given that UTR predictions in reference databases are not always precise, such observations should be interpreted cautiously. Expression analysis using an isoform-specific k-mer approach confirmed that identical isoforms can be differentially regulated. These findings suggest that, beyond expanding protein diversity, alternative splicing can also generate redundant isoforms that are differentially expressed at the RNA level, indicating potential regulatory roles. By elucidating the structural and regulatory factors contributing to the formation and retention of identical isoforms, our study provides new insights into the evolutionary and functional significance of alternative splicing in plants.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7434-7448"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148040565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.","authors":"Alicia Mendoza, Isabella Simon, Sharmin Hasan","doi":"10.1007/s10528-026-11400-7","DOIUrl":"10.1007/s10528-026-11400-7","url":null,"abstract":"<p><p>Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.</p>","PeriodicalId":482,"journal":{"name":"Biochemical Genetics","volume":" ","pages":"7759-7786"},"PeriodicalIF":1.9,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148025610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}