Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line–competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBa c transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications.

Significance: Targeted gene knockout by editing specific loci in genome has revolutionized the field of functional genomics. Transcription activator-like effector nucleases (TALENs) are representative next-generation platforms for customized genomic editing in transgenic animals, as well as cultured cells in vitro. In this study, in combination with chicken primordial germ cell line with germ-line transmission capacity, we generated the ovalbumin gene knockout chickens by TALEN-mediated gene targeting. Our results extended the application of state-of-the-art TALEN technology from experimental animals to farm animals. As TALEN-mediated knockout chickens are genetically modified but nontransgenic, without genomic integration of any exogenous transgenes, specific genomic editing with TALENs could expedite the generation of genetically engineered chickens for agriculturally practical applications as well as for model animals.

Long-term maintenance of avian primordial germ cells (PGCs) in vitro has tremendous potential because it can be used to deepen our understanding of the biology of PGCs. A transgenic bioreactor based on the unique migration of PGCs toward the recipients' sex cord via the bloodstream and thereby creating a germline chimeric bird has many potential applications. However, the growth factors and the signaling pathway essential for inducing proliferation of chicken PGCs are unknown. Therefore, we conducted this study to investigate the effects of various combinations of growth factors on the survival and proliferation of PGCs under feeder-free conditions. We observed proliferation of PGCs in media containing bFGF. Subsequent characterization confirmed that the cultured PGCs maintained expression of PGC-specific markers, telomerase activity, normal migrational activity, and germline transmission. We also found that bFGF activates the mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/ERK) signaling. Also, the expression of 133 transcripts was reversibly altered by bFGF withdrawal. Our results demonstrate that chicken PGCs can be maintained in vitro without any differentiation or dedifferentiation in feeder free culture conditions, and subsequent analysis revealed that bFGF is one of the key factors that enable proliferation of chicken PGCs via MEK/ERK signaling regulating downstream genes that may be important for PGC proliferation and survival.

In the avian species, germline chimera production could be possible by transfer of donor germ cells into the blood vessel of recipient embryos. This