Production of germline chimeric chickens, with high transmission rate of donor‐derived gametes, produced by transfer of primordial germ cells

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Abstract
Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13–15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14–15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor-derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor-derived offspring was 81% for three male chimeras (96% for one female chimera), and it was ∼3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor-derived offspring by 10% in resulting male chimeras. Male chimeras produced donor-derived offspring more frequently (∼3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor-derived offspring from the germline chimeras with increasing age. Male to female ratio of the offspring derived from the donor primordial germ cells did not deviate significantly in male and female chimeras, suggesting that primordial germ cells that have different sex from recipient embryos could not differentiate into functional gametes. The technique for primordial germ cell transfer employed in this experiment is simple to perform and resulted in the efficient production of germline chimeras with high transmission rates of donor-derived gametes. This system provides a powerful tool for avian embryo manipulation.