Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds

Abstract

In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10^-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.

Keywords: biodiversity; conservation; genome editing; poultry; primordial germ cell.

Fig. 1.

Germline transmission using layer sterile surrogate hosts. (A and B) Vantress heritage broiler PGCs labeled with GFP or TdTomato fluorescent reporter transposons. (C and D) Ovary from a DDX4 Z⁻ W hen at 8 wk posthatch injected with labeled PGCs. (E) GFP⁺ offspring from DDX4 Z⁻ W host hens. DDX4 Z⁻ W hosts were artificially inseminated with layer semen, and hatchlings were screened for fluorescence.

Fig. 2.

Principal component (PC) analysis of offspring from DDX4 Z⁻ W and ZW surrogate hosts. DNA samples were genotyped on a 66,000-SNP chip and analyzed for PCs. Three chicken breeds were analyzed: the Vantress heritage breed (blue), an independent pedigree broiler line (red), and the Hy-Line brown layer DDX4 surrogate host line (brown). Offspring (green and gray) from the DDX4 Z⁻ W hosts clustered between the Vantress breed and the Brown layer line. Offspring (black) from a DDX4 Z⁻W host inseminated with Vantress semen clustered with the Vantress breed chicken.

Fig. 3.

Pure offspring produced from DDX4 Z⁻ W hens using heritage broiler semen. (A) Fresh or (B) cryopreserved semen pooled from 3 adult males was used to fertilize a DDX4 Z⁻ W founder female. The pure line Vantress chick in B is shown surrounded by 2 control layer offspring.

Fig. 4.

Diagram of the reconstitution of a poultry breed using cryopreserved cells. Cryopreserved female rare breed PGCs introduced into a sterile surrogate host hen inseminated with frozen rare breed semen will produce “pure” rare breed offspring.

Fig. 5.

Total SNVs mapped in 8 clonal cell lines compared with somatic embryonic DNA. PGCs were cultured from individual embryos and then cultured clonally after being propagated 55 d in culture. PGC DNA was compared with somatic DNA from the original embryo. Lines 19 and 20, male PGCs; lines 70 and 81, female PGCs. A, B, and C suffixes indicate individual clonal populations derived from each PGC line.