Innovations in poultry reproduction using cryopreserved avian germ cells

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals.</p

Study on host immune responses in birds

内容の要約広島大学(Hiroshima University)博士(学術)Doctor of Philosophydoctora

Genome editing in chickens

Chicken (Gallus gallus) is a major protein source and an important model organism in the avian species. Although genome editing has enabled genetic modifications of various organisms and has a significant impact on academia and industry, its application to chickens has been comparatively delayed owing to difficulties in handling their one-cell fertilized eggs. Thus, researchers have attempted to produce genome-edited chickens using primordial germ cells (PGCs), which are precursor cells of sperm or eggs. Currently, genome-edited chickens can be produced with the development of avian biotechnologies, PGCs culture methods, and germline chimerism systems, in particular. In this review, we describe the current status of genome editing in chickens, including avian biotechnologies, with a primary focus on the achievements of Japanese researchers. In addition, we discuss the remaining issues and make suggestions for future research

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken

Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells

ABSTRACT: Activation of Wnt/β-catenin signaling supports the self-renewal of mouse embryonic stem cells. We aimed to understand the effects of Wnt signaling activation or inhibition on chicken embryonic stem cells (chESCs), as these effects are largely unknown. When the glycogen synthase kinase-3 β inhibitor CHIR99021—which activates Wnt signaling—was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related (NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B, and PRDM14) and germ cell (CVH and DAZL) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d. By contrast, when the Wnt signaling inhibitor XAV939 was added to the culture, dense and compact multipotent colonies (morphologically similar to mouse embryonic stem cell colonies) showing stable expression of pluripotency-related and germline markers were formed. The addition of XAV939 stabilized the proliferation of chESCs in the early stages of culture and promoted their establishment. Furthermore, these chESCs formed chimeras. In conclusion, functional chESCs can be stably cultured using Wnt signaling inhibitors. These findings suggest the importance of Wnt/β-catenin signaling in avian stem cells, offering valuable insights for applied research using chESCs