Health Hen Project Sample Map Revive & Restore

Phase One

Results of Heath Hen Genomic Study

Revive & Restore has sequenced the genomes of the Heath Hen and all three living species in its genusTympanuchus: the Greater Prairie Chicken (T. cupido pinnatus) and its subspecies the Attwater’s Prairie Chicken (T. c. attwateri); the Lesser Prairie Chicken (T. pallidicinctus), and two subspecies of Sharp-tailed Grouse, (T. phasianellus kennicoti) and (T. phasianellus jamesi). A total of 19 genomes were sequenced to decipher the exact relationship of the Heath Hen to its living relatives. Our high quality reference genome of the Greater Prairie Chicken, assembled by Dovetail Genomics, is now available on the National Center for Biotechnology Information’s public GenBank database.

Once thought to be a subspecies of Greater Prairie Chicken, the Heath Hen’s genome reveals that it is a distinct and unique species, despite its similarities to the Greater Prairie Chicken. Preliminary analysis shows that among the living species and subspecies, the Greater Prairie Chicken appears to be the closest genetic template for Heath Hen de-extinction.

These results prompted the initiation of Phase 2 preparations in 2015, with the establishment of Revive & Restore’s own Greater Prairie Chicken flock with project partner Grouse Park, owned and operated by Dan Snyder (pictured below).

Heath Hen graph Revive & Restore

As the preparatory work for Phase 2 began, the discovery stages of genome research ramped up: nearly 20,000 genes of the Heath Hen’s genome are being identified and compared to the Greater Prairie Chicken. In the coming years, our team, lead by Dr. Jeff Johnson of the University of Northern Texas, will be identifying candidate genes that may support unique adaptations to the Heath Hen’s unknown New England life history. These genes will form the basis for reviving the species; these genes will be edited into the genomes of Greater Prairie Chickens.

Grouse Park

Dan feeding animals Revive and Restore

Dan Snyder at Grouse Park (Idaho)

Nest with Eggs Revive & Restore

Greater Prairie Chicken Eggs, Grouse Park

Phase Two

In Vitro Research

Early Phase 2 research presented the first scientific challenges on the path to successful de-extinction as Revive & Restore’s partners began researching and developing the techniques necessary to breed genome edited Greater Prairie Chickens.

In order for the revival of the Heath Hen to be possible, engineered cells in a petri dish must develop into live chicks. This must be accomplished by using a process known as germ-line transmission, where the primordial germ cells (PGCs) of an early embryo are edited, grown in cultures, and then reproductively transmitted through surrogate host parents to generate live, engineered birds. For Heath Hen de-extinction, the desired surrogate parent to host Heath Hen germ-lines is the domestic chicken.

Research Steps

Heath Hen Phase 2 Revive & Restore

Towards this goal, our project partners accomplished a large amount of work from March to December 2016, beginning with Greater Prairie Chicken breeding at Grouse Park.

Over the course of the two-month breeding season, Grouse Park provided 72 eggs for germ-cell culturing experiments at Crystal Bioscience’s laboratories. An additional six Greater Prairie Chicken chicks were hatched and hand-raised to adulthood for further reproductive technology development in the coming years.

From the eggs provided by Grouse Park, Crystal Biosciences researchers were able to establish five viable cell lines from a series of experimental culture conditions. Early in vitro analyses of gene expression revealed that the cell lines were germ-cells, our target cell type. This discovery generated a high level of excitement for our team.

The next step was to inject the germ-cells into developing domestic chicken embryos: the first step to creating the surrogate germ-line chimeric parents. A particular developmental stage of germ-cells is required for germ-line transmission to work, which is the earliest stage known as “primordial germ cells.” Only in the primordial state will the germ-cells migrate through the embryonic blood stream and integrate into the reproductive system of the developing chick.

Green Glowing Cells Revive & Restore

Pictured Above: Greater Prairie Chicken germ-cells, fluorescing green thanks to the integration of a GFP marker gene. These are the world’s first genetically engineered Greater Prairie Chicken cells.

But the replicate tests in domestic chicken embryos all failed, revealing that while the cells were definitely germ cells, they were not the primordial germ cells needed for recreating Heath Hens. The germ cells had developed beyond the early primordial state, meaning they were no longer capable of migrating through embryonic tissues. The results were a sobering reminder of the nature of working on cutting edge science.

Despite this disappointing news, the research conducted in 2016 was invaluable:

  • We developed standard operating procedures for working with Greater Prairie Chicken eggs and embryos from the aviary to the laboratory.
  • We identified the growth factors necessary to keep Greater Prairie Chicken germ cells alive in culture. These growth factors, when mixed in the right culture medium, work very well with late stage germ cells, and will likely work well with earlier stage germ cells after some minor adjustments.
  • Our partner, Dan Snyder of Grouse Park, clearly demonstrated that a small flock of Greater Prairie Chickens can produce the ideal amount of fertile eggs for germ-line transmission research. Grouse Park produced more eggs than needed, allowing Dan to incubate extra eggs and hand raise several Greater Prairie Chicken offspring that will be exceptionally tame. These birds will be crucial to the future stages of germ-line transmission research that require extensive handling of captive birds.

The knowledge gained and resources developed in 2016 will allow us to refine the process of isolating germ cells, bringing Revive & Restore closer to achieving our goal of hatching Greater Prairie Chicken offspring via germ-line transmission, the first major step toward hatching a new generation of Heath Hens. If this work is successful, it will be a first for conservation and de-extinction efforts.

Next Steps

We are currently raising funds for the next part of our Phase 2 research: resuming germ-line transmission research in spring 2018, drawing upon lessons learned from our previous research and making the appropriate modifications. As the necessary milestones to revive Heath Hens are accomplished, we intend to develop the habitat restoration research and assessments that will be vital to move Heath Hens from habitat enclosures to free ranging flocks. The process of reintroduction will require considerable regulatory dialogue at community, state, and federal levels, which could set significant precedent for de-extinction and genetic rescue practices. Revive & Restore’s team of advisors and collaborators are passionately dedicated to facilitating community dialogue for the advancement of this ambitious restoration venture.