Search Results (29)

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play crucial roles in the regulation of gene expression. In poultry, miRNAs have emerged as significant regulators of various biological processes, such as growth, development, immune response, and disease resistance in humans, plants, and animals. Recent studies have identified numerous miRNAs in different poultry species (chickens, geese, turkeys, quails, and ducks) and have elucidated their roles in various economically important traits in these species. This review aims to provide a comprehensive overview of the progress made in understanding the functions and mechanisms of miRNAs in poultry as well as the challenges faced in this field. Firstly, an overview of progress in the identification and validation of miRNAs in each species is provided. Then, specific roles of miRNAs in different classes of phenotypic traits are presented. Finally, the challenges and impacts of key technologies and methods, including artificial intelligence, genome editing, and single-cell sequencing, are discussed. From state-of-the-art research on miRNA functions across different trait classes to up-to-date technology trends, this review provides a comprehensive picture of miRNAs in poultry, which might open up new research and application possibilities. Full article

Avian leukosis virus subgroup J (ALV-J), an α-retrovirus, mediates infection by binding to the host-specific receptor chNHE1 (chicken sodium–hydrogen exchanger type 1), leading to immunosuppression and tumorigenesis, which severely threatens the sustainable development of the poultry industry. Studies have shown that the tryptophan residue at position 38 (W38) of the chNHE1 protein is the critical site for ALV-J infection. In this study, we employed the CRISPR/Cas9 system to construct a lentiviral vector targeting the W38 site of chNHE1, transfected it into chicken primordial germ cells (PGCs), and validated its antiviral efficacy through ALV-J infection assays, successfully establishing an in vitro gene-editing system for chicken PGCs. The constructed dual lentiviral vector efficiently targeted the W38 site. PGCs isolated from 5.5- to 7-day-old chicken embryos were suitable for in vitro gene editing. Stable fluorescence expression was observed within 24–72 h post-transfection, confirming high transfection efficiency. ALV-J challenge tests demonstrated that no viral env gene expression was detected in transfected PGCs at 48 h or 72 h post-infection, while high env expression was observed in control groups. After 7 days of infection, p27 antigen ELISA tests were negative in transfected groups but positive in controls, indicating that W38-deleted PGCs exhibited strong resistance to ALV-J. This study successfully generated ALV-J-resistant gene-edited PGCs using CRISPR/Cas9 technology, providing a novel strategy for disease-resistant poultry breeding and advancing avian gene-editing applications. Full article

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology. Full article

Chicken semen cryopreservation is crucial for utilizing high-quality cockerel genetics, but semen is highly sensitive to cryoinjury, leading to poor preservation outcomes. This study aimed to establish a theoretical foundation for selecting cockerels for semen cryopreservation through serum testing and to improve semen quality via DNA methylation editing. Semen and serum samples were collected from 102 Xiaoshan cockerels, with semen cryopreserved and thawed following standardized protocols. Post-thaw semen quality and serum testosterone (T) levels were assessed. Eight cockerels were selected based on motile sperm quality, and whole-genome bisulfite sequencing (WGBS) was used to analyze sperm DNA methylation. The results showed a significant positive correlation between serum T levels and sperm motility. There were notable differences in sperm motility and serum T levels between high-quality and low-quality semen groups but no differences in estradiol (E₂), superoxide dismutase (SOD), or glutathione peroxidase (GSH-Px) levels. A total of 217 differentially methylated regions (DMRs) and 116 differentially methylated genes (DMGs) were identified. Key genes such as PRKACB (protein kinase, cAMP-dependent, catalytic, beta) and ACSL1 (long-chain-fatty-acid--CoA ligase 1) were associated with sperm motility. These findings provide important insights for improving semen cryopreservation and contribute to breeding practices and the development of cryoprotectants. Full article

The successful application of primordial germ cells (PGCs) is an ideal method for generating gene-edited birds. However, barriers to efficient DNA transfection in PGCs lead to low transfection efficiency, limiting the generation of genetically modified chickens. The current study utilized chemical transfection and electroporation methods to determine the optimal transfection conditions for the PGC line under feeder- and serum-free medium. Among the tested methods, the Lonza electroporation system exhibited the highest transduction efficiency, with a previously unreported rate of 71.13 ± 1.26%. Optimal transfection conditions were achieved using 4 µg of DNA and 100 µL of Entranster^TM-E in 1 × 10⁶ PGCs. Furthermore, the optimal electroporation conditions resulted in low cell death and normal expression of pluripotency-related genes, highlighting the low cytotoxicity. The resulting electroporation models were then used to deliver the enhanced green fluorescent protein (EGFP) gene to the Z chromosome with a Cas9-gRNA plasmid, achieving a 7-day insertion efficiency of 14.63 ± 1.07%. Our study highlights the vast potential of electroporation technology for the transfection of PGCs. Full article

The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation. Full article

Many local chicken breeds are rapidly declining and even facing extinction due to a variety of factors such as indiscriminate crossbreeding, climate fluctuations, epidemic outbreak, and environmental changes. Developing effective preservation strategies is important to address this situation. The special reproductive characteristics and gamete morphology of chickens pose challenges for preserving genetic heritage through the cryopreservation of genetic materials. Currently, gonad and primordial germ-cell cryopreservation and transplantation are the most promising approaches, especially for the genetic information in the W chromosome of female birds. The study of establishing sterile recipients is crucial for increasing the efficiency of the colonization of transplanted donor tissues and cells. Several classical methods, including ovariectomy and testectomy, busulfan, and irradiation, have been employed to deplete endogenous germ cells in recipient males before transplantation. These methods rely on the toxicity of chemical reagents and physical stimulation to kill germ cells. Recent advances in gene-editing technology have introduced sterile hosts via the knocking out of genes relevant to germ cells’ development. This review explores state-of-the-art technologies for preparing infertile avian recipients (mainly chickens) and aims to provide guidance for the conservation of poultry genetic material and breed restoration. Full article

Since the outbreak of the H9N2/Y439 avian influenza virus in 1996, the Korean poultry industry has incurred severe economic losses. A novel possibly zoonotic H9N2 virus from the Y280-like lineage (H9N2/Y280) has been prevalent in Korea since June 2020, posing a threat to the poultry sector. Rapid mutation of influenza viruses urges the development of effective vaccines against newly generated strains. Thus, we engineered a recombinant virus rHVT/Y280 to combat H9N2/Y280. We integrated the hemagglutinin (HA) gene of the H9N2/Y280 strain into the US2 region of the herpesvirus of turkeys (HVT) Fc126 vaccine strain, utilizing CRISPR/Cas9 gene-editing technology. The successful construction of rHVT/Y280 was confirmed by polymerase chain reaction and sequencing, followed by efficacy evaluation. Four-day-old specific pathogen-free chickens received the rHVT/Y280 vaccine and were challenged with the H9N2/Y280 strain A21-MRA-003 at 3 weeks post-vaccination. In 5 days, there were no gross lesions among the vaccinated chickens. The rHVT/Y280 vaccine induced strong humoral immunity and markedly reduced virus shedding, achieving 100% inhibition of virus recovery in the cecal tonsil and significantly lowering tissue viral load. Thus, HVT vector vaccines expressing HA can be used for protecting poultry against H9N2/Y280. The induction of humoral immunity by live vaccines is vital in such cases. In summary, the recombinant virus rHVT/Y280 is a promising vaccine candidate for the protection of chickens against the H9N2/Y280. Full article

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is an extremely contagious immunosuppressive disease that causes major losses for the poultry industry worldwide. Recently, the novel variant IBDV (G2d) has been highly prevalent in Korea, but the current vaccines against this very virulent IBDV have limited efficacy against this novel variant. To develop a vaccine against this variant IBDV, a recombinant virus designated rHVT-VP2 was constructed by inserting the IBDV (G2d) VP2 gene into herpesvirus of turkeys (HVT) using CRISPR/Cas9 gene-editing technology. The PCR and sequencing results obtained showed that the recombinant virus rHVT-VP2 was successfully constructed. Vaccination with rHVT-VP2 generated IBDV-specific antibodies in specific pathogen-free chickens starting from 2 weeks post-immunization. Seven days after the challenge, the autopsy results showed that the bursa atrophy rates of the rHVT-VP2, HVT, vaccine A, and positive control groups were 0%, 100%, 60%, and 100%, respectively, and the BBIX values were 1.07 ± 0.22, 0.27 ± 0.05, 0.64 ± 0.33, and 0.32 ± 0.06, respectively. These results indicate that rHVT-VP2 can provide 100% protection against a challenge with the IBDV (G2d), whereas vaccine A only provides partial protection. In conclusion, vaccination with the recombinant virus rHVT-VP2 can provide chickens with effective protection against variant IBDV (G2d). Full article

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases. Full article

Perilipin 1 (PLIN1) is one of the most abundant lipid droplet-related proteins on the surface of adipocytes. Our previous results showed that PLIN1 plays an important role in chicken lipid metabolism. To further reveal the role of PLIN1 in the growth and development of adipocytes, a chicken preadipocyte line with a PLIN1 gene knockout was established by the CRISPR/Cas9 gene editing technique, and the effects of the PLIN1 gene on the proliferation, apoptosis, differentiation and lipolysis of chicken preadipocytes were detected. The results showed that the CRISPR/Cas9 system effectively mediated knockout of the PLIN1 gene. After the deletion of PLIN1, the differentiation ability and early apoptotic activity of chicken preadipocytes decreased, and their proliferation ability increased. Moreover, knockout of PLIN1 promoted chicken preadipocyte lipolysis under basal conditions and inhibited chicken preadipocyte lipolysis under hormone stimulation. Taken together, our results inferred that PLIN1 plays a regulatory role in the process of proliferation, apoptosis, differentiation and lipolysis of chicken preadipocytes. Full article

Avian leukosis virus (ALV) induces B-cell lymphomas and other malignancies in chickens through insertional activation of oncogenes, and c-myc activation has been commonly identified in ALV-induced tumors. Using ALV-transformed B-lymphoma-derived HP45 cell line, we applied in situ CRISPR-Cas9 editing of integrated proviral long terminal repeat (LTR) to examine the effects on gene expression and cell proliferation. Targeted deletion of LTR resulted in significant reduction in expression of a number of LTR-regulated genes including c-myc. LTR deletion also induced apoptosis of HP45 cells, affecting their proliferation, demonstrating the significance of LTR-mediated regulation of critical genes. Compared to the global effects on expression and functions of multiple genes in LTR-deleted cells, deletion of c-myc had a major effect on the HP45 cells proliferation with the phenotype similar to the LTR deletion, demonstrating the significance of c-myc expression in ALV-induced lymphomagenesis. Overall, our studies have not only shown the potential of targeted editing of the LTR for the global inhibition of retrovirus-induced transformation, but also have provided insights into the roles of LTR-regulated genes in ALV-induced neoplastic transformation. Full article

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses. Full article

Background: EGFR mutations are present in approximately 15–50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors’ activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI₅₀ score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI₅₀ < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines. Full article

Fragile X encompasses a range of genetic conditions, all of which result as a function of changes within the FMR1 gene and abnormal production and/or expression of the FMR1 gene products. Individuals with Fragile X syndrome (FXS), the most common heritable form of intellectual disability, have a full-mutation sequence (>200 CGG repeats) which brings about transcriptional silencing of FMR1 and loss of FMR protein (FMRP). Despite considerable progress in our understanding of FXS, safe, effective, and reliable treatments that either prevent or reduce the severity of the FXS phenotype have not been approved. While current FXS animal models contribute their own unique understanding to the molecular, cellular, physiological, and behavioral deficits associated with FXS, no single animal model is able to fully recreate the FXS phenotype. This review will describe the status and rationale in the development, validation, and utility of three emerging animal model systems for FXS, namely the nonhuman primate (NHP), Mongolian gerbil, and chicken. These developing animal models will provide a sophisticated resource in which the deficits in complex functions of perception, action, and cognition in the human disorder are accurately reflected and aid in the successful translation of novel therapeutics and interventions to the clinic setting. Full article