Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds Mark E. Woodcocka,1, Almas A. Gheyasa,1, Andrew S. Masona, Sunil Nandia, Lorna Taylora, Adrian Shermana, Jacqueline Smitha, Dave W. Burtb, Rachel Hawkenc, and Michael J. McGrewa,2 aDivision of Functional Genetics and Development, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, EH25 9RG Midlothian, United Kingdom; bOffice of Deputy Vice-Chancellor (Research), The University of Queensland, Brisbane, QLD 4072, Australia; and cCobb- Europe, CO7 7QR Colchester, United Kingdom Edited by Martin M. Matzuk, Baylor College of Medicine, Houston, TX, and approved September 6, 2019 (received for review April 15, 2019) In macrolecithal species, cryopreservation of the oocyte and in vertebrate species and during in vitro culture (17). Mammalian zygote is not possible due to the large size and quantity of lipid embryonic stem cells also have lower mutation rates in vitro deposited within the egg. For birds, this signifies that cryopreserv- (∼100-fold)whencomparedwithsomaticcells(18). ing and regenerating a species from frozen cellular material are Chicken is one of the few species from which PGCs can easily currently technically unfeasible. Diploid primordial germ cells be propagated in vitro to increase cell number using a defined (PGCs) are a potential means to freeze down the entire genome medium (19). For chicken, PGCs from a single embryo can be and reconstitute an avian species from frozen material. Here, we expanded in vitro to >100,000 cells within 4 wk and subsequently examine the use of genetically engineered (GE) sterile female layer cryopreserved. Chicken PGCs can also be genetically modified chicken as surrogate hosts for the transplantation of cryopre- during in vitro culture (9, 20–24). After thawing, PGCs transfer served avian PGCs from rare heritage breeds of chicken. We first into the embryonic vascular system of “surrogate host” embryos, amplified PGC numbers in culture before cryopreservation and where they migrate to the forming gonads and will differentiate subsequent transplantation into host GE embryos. We found that into functional gametes in the adult host (10, 25, 26). The adult all hatched offspring from the chimera GE hens were derived from surrogate host chickens are subsequently bred to generate off- the donor rare heritage breed broiler PGCs, and using cryopre- spring, some of which derive from the exogenous donor PGCs. SCIENCES served semen, we were able to produce pure offspring. Mea- Layer breeds of chicken, which have been selected for egg pro- AGRICULTURAL surement of the mutation rate of PGCs in culture revealed that duction, can be used as surrogate hosts for transplanted germ 2.7 × 10−10 de novo single-nucleotide variants (SNVs) were gener- cells from other breeds of less fertile chicken and conceivably, ated per cell division, which is comparable with other stem cell from other avian species (27–29). A major constraint to the use lineages. We also found that endogenous avian leukosis virus of this system is that the transmission rate from exogenous PGCs (ALV) retroviral insertions were not mobilized during in vitro prop- injected into layer chicken embryos can vary greatly between agation. Taken together, these results show that mutation rates individual surrogate host animals and between the different are no higher than normal stem cells, essential if we are to con- chicken breeds used as surrogate hosts (25, 30). To circumvent serve avian breeds. Thus, GE sterile avian surrogate hosts provide this problem, chemical and physical methods have been used to a viable platform to conserve and regenerate avian species using ablate the endogenous germ cells of the surrogate host and have cryopreserved PGCs. Significance conservation | biodiversity | primordial germ cell | poultry | genome editing In the fields of conservation biology and sustainable agricul- ture, the ability to cryopreserve and revive animal species is ryopreservation of the oocyte and zygote is not possible in paramount to efforts to preserve genetic diversity. An in- Cmacrolecithal species, such as birds and fish, due to the large novative approach is to use sterile surrogate host animals for amount of lipid deposited in the female oocyte (1–3). For avian the transplantation of reproductive germ cells from rare/ species, this signifies that, to conserve a breed of interest, birds endangered animals. This technology has previously been uti- must be maintained as extant breeding populations, which places lized in mammals but is of particular importance for animals them in danger to losses in biodiversity caused by population with lipid-filled zygotes/embryos, such as fish and birds, which fluctuations and to the constant threat of extinction. The em- render cryopreservation techniques inefficient. We demon- bryonic diploid reproductive germ cells from avian species offer strate that the female chicken rendered sterile using genome an alternative means to cryopreserve the entire genotype of the editing technology can be used as a surrogate host for trans- germplasm (reproductive cells) from which a pure breeding planted cryopreserved germ cells and only lay eggs of the population could be entirely reconstituted at a later date. This is transplanted rare chicken breed. Our results suggest a way to a current research objective being pursued for both avian and preserve the biodiversity of bird species. fish species as a way to safeguard the genetic diversity of both farmed and rare/endangered breeds and species (2, 4, 5). The Author contributions: M.E.W., A.A.G., A.S.M., S.N., D.W.B., R.H., and M.J.M. designed research; M.E.W., A.A.G., A.S.M., S.N., L.T., A.S., R.H., and M.J.M. performed research; embryonic or primordial germ cells (PGCs) can be cryopreserved A.S.M. contributed new reagents/analytic tools; M.E.W., A.A.G., A.S.M., S.N., L.T., A.S., directly, or since their number is low in the early embryo (50 to J.S., D.W.B., and M.J.M. analyzed data; and M.E.W., A.A.G., A.S.M., S.N., L.T., J.S., 150 cells) (6–8), PGCs from a few select species can be propagated D.W.B., R.H., and M.J.M. wrote the paper. in culture to increase their number before cryopreservation (9–11). The authors declare no competing interest. The germ cell lineage is also believed to safeguard genetic This article is a PNAS Direct Submission. information by having both high levels of homologous recombi- Published under the PNAS license. nation and enzymes for DNA repair and by initiating pro- 1M.E.W. and A.A.G. contributed equally to this work. grammed cell death when double-strand breaks are formed (12– 2To whom correspondence may be addressed. Email: [email protected]. 16). The mutation rate of vertebrate germ cells in culture has not This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been measured but is thought to be low in comparison with so- 1073/pnas.1906316116/-/DCSupplemental. matic cell lineages, as intergenerational mutation rates are low www.pnas.org/cgi/doi/10.1073/pnas.1906316116 PNAS Latest Articles | 1of8 Downloaded by guest on September 29, 2021 been shown to increase the transmission of donor cell genetics. PGC cultures for all of these breeds were subsequently cryopreserved However, these agents are highly toxic to both the developing in vials containing 50,000 cells. PGCs survived cryopreservation surrogate host embryo and the mature host animal (10, 31–33). well and proliferated robustly when recultured after thawing (SI Alternatively, the genetic ablation of a gene required for germ Appendix,Fig.S1). cell development has been used to eliminate the endogenous germ cells in both mammalian and fish species (34–38). These geneti- Germline Transmission Using Female Sterile Surrogate Hosts. Mouse cally sterile surrogate hosts were subsequently shown to efficiently embryonic stem cells and chicken PGCs have been shown to lose generate offspring deriving from transplanted exogenous germ cells. germline competence after extended periods in culture (42). To We recently used genome editors to disrupt the chicken DDX4 measure both germline transmission and loss of germline com- (Vasa) gene, which is located on the Z sex chromosome in bird petence during in vitro propagation, we continuously propagated species (39). In birds, males are the homogametic sex containing the heritage broiler breed PGCs in vitro for a total time of 3 or ZZ sex chromosomes, whereas females are the heterogametic 6 mo before cryopreservation. We genetically labeled individual DDX4 − sex containing ZW sex chromosomes. In Z W mutant cultures of male or female PGCs with a fluorescent marker using females, we observed that PGCs were reduced in number in the piggyBac transposons containing either GFP (green; 3 mo) or developing embryo and entirely absent in the posthatch ovary, tdTomato (red; 6 mo) fluorescent reporter genes early on during leading to ovarian atrophy and a failure to lay eggs. Accordingly, − this culture period. Using labeled PGCs (Fig. 1 A and B) enables the transfer of exogenous donor germ cells into DDX4 Z W us to lineage trace the germ cells during embryonic development host females during embryonic development may rescue oocyte and to easily identify offspring deriving from the PGCs after formation and restore egg production. Subsequent insemination of the DDX4 surrogate host with cryopreserved semen from the injection into surrogate host embryos. Aliquots of frozen, labeled same donor breed would permit the complete reconstitution of the breed from frozen cellular material. In this study, we demonstrate the reconstitution of a chicken breed from frozen cellular material. PGCs were first isolated from several rare traditional breeds of chicken and propagated in vitro to increase germ cell numbers before cryopreservation.