Chickens are an ideal animal model study tool for developmental biology, and a farm animal with excellent productivity. Researchers have therefore long sought to establish chicken embryonic stem cells (cESCs) to enable the creation of genetically modified chickens. Here, we derived novel cESCs from chicken blastodermal cells (CBCs) cultured with chicken leukemia inhibitory factor (chLIF). These cESCs have the capacity for long-term ( days or more) successive subculture and express both chicken Nanog (chNanog) and chicken vasa homolog (Cvh) mRNAs and proteins. The cESCs showed a capacity for chimeric formation during a transplant experiment that used a fertilized egg. Transfer of the enhanced green fluorescent protein (EGFP) gene to cESCs enabled green fluorescence to be observed among primordial germ cells (PGCs). These results indicate that novel cESCs should have the capacity to di erentiate into germ cells.: chicken embryonic stem cells, chicken Nanog, chicken vasa homolog, leukemia inhibitory factor, primordial germ cells et al., et al.,

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

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 knockin 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.

Background Birds harbor specific sex determination and differentiation mechanisms. Although the molecular mechanisms associated with sex determination in somatic cells have been elucidated, those for germ cells remain unclear. Results Here, we characterized the chicken forkhead box L3 (foxl3)‐like gene as a sex‐determination factor in sexually indifferent medaka germline stem cells. The foxl3‐like gene was cloned by rapid amplification of cDNA ends, and the nucleotide sequence was analyzed. The deduced amino acid sequence was compared with FOXL3 sequences from other species, revealing low identity and similarity scores. Expression analysis of foxl3‐like mRNA during gonadogenesis showed female left‐gonad‐specific temporal expression in an egg incubated from 10 to 16 days, as well as low general expression in certain hatched female chicken organs. Moreover, the amino acid sequence deduced for the FOXL3‐like protein displayed low identity with medaka FOXL3, with the FOXL3‐like protein specifically localized in the oogonia, whereas medaka FOXL3 was found in sexually indifferent germline stem cells. Furthermore, the timing of expression differed between the foxl3‐like gene and that of medaka foxl3. Conclusions These results suggest that chicken FOXL3‐like protein and medaka FOXL3 differ in terms of their functions as female sex‐determination factors.

We would like to thank Editage (www.editage.jp) for English language editing.