[1S06e] 15. Advanced Cell and Cell Culture Engineering for Biopharmaceuticals and Functional Food - Part 3

6:10 PM - 6:30 PM JST(9:10 AM - 9:30 AM UTC)

[1ACCE-15] New transgenic chicken resources in Avian Bioscience Research Center, Nagoya University for biopharmaceutical and food production

○Ken-ichi Nishijima¹, Yuya Okuzaki¹, Ren Liang¹ (1. Nagoya University (Japan))

Keywords:

Chicken,Transgenic,Resources,Quail,Protein production

[Background]Continuous breeding of chickens and quail in the poultry industry has greatly improved productivity and cost-effectiveness, leading to the global dominance of a limited number of commercial breeds. In contrast, other strains with specific traits represent valuable genetic resources for the future improvement of poultry lines and also serve as important model organisms in life sciences, bridging the evolutionary gap between mammals and other vertebrates. The Avian Bioscience Research Center (ABRC) at Nagoya University maintains and distributes chicken and quail resources as a core facility for avian bioresources within the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. These resources include lines established through traditional breeding methods, such as inbred strains—rare in birds—as well as mutant and disease model strains. Some strains may also serve as models for poultry diseases. For example, a strain exhibiting muscular dystrophy shows symptoms partly resembling the muscle stiffness observed in broiler meat associated with rapid growth, known as Woody Breast. [Purpose]Recent advances in genetic modification technologies have enabled the establishment of various transgenic and genome-edited chicken lines. At ABRC, we maintain several transgenic chickens and quails useful for both basic and applied poultry sciences. These include fluorescent reporter lines ubiquitously expressing EGFP, cell-type-specific fluorescent reporter lines (e.g., germ cells, limb bud mesenchyme, and apical ectodermal ridge cells), CRISPR/Cas9-expressing lines enabling in vivo gene knockout, and lines designed for the production of biologically active substances in eggs. These developments rely on techniques for the long-term culture of chicken primordial germ cells (PGCs), embryonic cells that give rise to both sperm and oocytes. In this study, we attempted to generate new transgenic chicken lines using cultured PGCs derived from White Leghorn. [Method]PGCs were obtained from the M/O strain of White Leghorn maintained at ABRC and cultured in vitro using a medium reported by Whyte et al. (2015) with minor modifications. Cells were transfected using Lipofectamine 2000 and selected based on drug resistance or reporter expression. Established PGC lines were transplanted into the bloodstream of 2.5-day embryos and incubated until hatch to obtain germline-chimeric chickens. After sexual maturation, roosters were mated with non-transgenic hens, and offspring were screened for the presence of the transgene by PCR analysis of genomic DNA. [Results]An expression construct for CRISPR interference (CRISPRi) was introduced into PGCs. Suppression of target gene expression was confirmed by the additional introduction of guide RNAs. To obtain CRISPRi transgenic chickens, the modified PGCs were injected into recipient embryos. By mating germline-chimeric roosters, we successfully obtained transgenic chickens with high efficiency, reaching up to 43% germline transmission. [Consideration]The established CRISPRi chickens provide a useful tool for investigating enhancer function and gene expression regulation in vivo. [Conclusion]These results demonstrate that transgenic chickens can be efficiently generated using cultured PGCs, providing a powerful platform for functional genomics and applied poultry research.