Suchergebnisse

Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells (PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds, including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs. Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A3A2033826).

Abbreviations CRDs: cysteine rich domains; ALV: avian leukosis virus; tvb: tumor virus locus B; tva: tumor virus locus A; tvc: tumor virus locus C; W38: tryptophan 38; CRISPR/ Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated; DMEM: Dulbecco's minimum essential medium; FBS: fetal bovine serum; ABAM: antibiotic–antimycotic; WL: White Leghorn; CEF: chicken embryonic fbroblast; EDTA: ethylenediaminetetraacetic acid; dpi: days postinfection; SAS: the statistical analysis system; ANOVA: analysis of variance; WT: wild type; GFP: green fuorescent protein; TNFRSF10B: tumor necrosis factor receptor superfamily member 10B; DD: death domain; PAM: protospacer adjacent motif; LTR: long terminal repeat; RCAS: replication-competent ALV LTR with a splice acceptor; HIV: human immunodefciency virus; EIAV: equine infectious anemia virus; FIV: feline immunodefciency virus; RABS: rabies virus; HVEM: herpes virus entry mediator; LDLR: low-density lipoprotein receptor; PGC: primordial germ cell; gRNA: guide RNA. ; Abstract Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection. ; This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A3A2033826). Authors also acknowledge the funding support from the Royal Society International Professorships.

Background Acquisition of pluripotency by transcriptional regulatory factors is an initial developmental event that is required for regulation of cell fate and lineage specification during early embryonic development. The evolutionarily conserved core transcriptional factors regulating the pluripotency network in fishes, amphibians, and mammals have been elucidated. There are also species-specific maternally inherited transcriptional factors and their intricate transcriptional networks important in the acquisition of pluripotency. In avian species, however, the core transcriptional network that governs the acquisition of pluripotency during early embryonic development is not well understood. Results We found that chicken NANOG (cNANOG) was expressed in the stages between the pre-ovulatory follicle and oocyte and was continuously detected in Eyal-Giladi and Kochav stage I (EGK.I) to X. However, cPOUV was not expressed during folliculogenesis, but began to be detectable between EGK.V and VI. Unexpectedly, cSOX2 could not be detected during folliculogenesis and intrauterine embryonic development. Instead of cSOX2, cSOX3 was maternally inherited and continuously expressed during chicken intrauterine development. In addition, we found that the pluripotency-related genes such as cENS-1, cKIT, cLIN28A, cMYC, cPRDM14, and cSALL4 began to be dramatically upregulated between EGK.VI and VIII. Conclusion These results suggest that chickens have a unique pluripotent circuitry since maternally inherited cNANOG and cSOX3 may play an important role in the initial acquisition of pluripotency. Moreover, the acquisition of pluripotency in chicken embryos occurs at around EGK.VI to VIII. ; This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2015R1A3A2033826) and the International Research & Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning of Korea (NRF-2016K1A3A1A21005676).