THE BLACK-FOOTED FERRET PROJECT
Revive & Restore was awarded its first federal grant from the U.S. Fish and Wildlife Service’s Recovery Challenge Fund for Endangered Species Conservation. This funding will go towards conducting foundational research in the use of synthetic biology approaches to dealing with the lethal sylvatic plague, which is the most serious challenge facing wild black-footed ferret populations. This is the first USFWS grant to explore the potential for cutting edge gene-editing techniques to help endangered species.
Today all black-footed ferrets released to the wild are vaccinated against the bacterium that causes plague, but their wild-born offspring do not inherit that immunity – meaning conservationists must capture and vaccinate wild-born ferrets to combat plague outbreaks. Any ferret they miss will die if exposed to plague. One of our newest projects is starting with domestic ferrets to develop and test a genetic vaccine that could be inherited from parent to offspring, which if successful would not only help black-footed ferrets but could be applied to many species threatened with invasive diseases. The hope for this work is that one-day black-footed ferrets may thrive in the wild without human intervention.
SPRING 2022 UPDATE
Elizabeth Ann, the world’s first cloned black-footed ferret, reached sexual maturity in the Spring of 2022 and as expected went into estrus at 16 months of age. However, during an artificial insemination attempt, it was discovered that Elizabeth Ann developed a uterine condition, hydrometra, that prevented her from being able to breed this year. This condition occurs in many natural-born mammals and has caused the death of black-footed ferrets in the past. Fortunately, the condition was found early, and for Elizabeth Ann’s safety, an emergency ovariohysterectomy was performed.
Elizabeth Ann has made a full recovery from surgery and is in good health. She continues to be as feisty as ever.
Revive & Restore and our partners announce their success in bringing Elizabeth Ann to the world. More milestones in cloning and sylvatic plague resistance are planned for 2021—and beyond.
This photo of Elizabeth Ann and the top photo of the Black-footed ferret courtesy of USFWS.
TRANSGENIC MICE ARE BORN
In January 2020, the first genetically engineered mice for the plague study were born at Texas A&M Institute for Genomic Medicine. These mice carry the gene for the plague-specific antibody that enables an early and effective immune response. They should exhibit innate resistance to sylvatic plague. If successful, the plan is to adapt this methodology for Black-footed ferrets.
INTERSPECIES PREGNANCY IS TESTED
Project partners ViaGen Pets and Marshall BioResources tested and confirmed the viability of interspecies pregnancy using test Black-footed ferret cell lines and domestic ferret surrogate mothers. The pre-parturition analyses appeared promising, which encouraged the USFWS to proceed with bringing cloned embryos to term in 2020.
CLONING IS PERFORMED
In November, ViaGen used the 32-year-old Willa cell line to develop cloned embryos. These were implanted into a domestic surrogate mother. Mid gestation, when the pregnancy was deemed safe, the mother was transferred to the National Black-footed Ferret Conservation Center. On December 10th, “Elizabeth Ann” became the first successfully cloned Black-footed ferret to be born. She is also the first endangered species native to the US to be cloned.
CLONING RESEARCH BEGINS
In 2019, concerted laboratory work supported through our Catalyst Science Fund began. Cloned Black-footed ferret embryos were successfully created in vitro, which showed that domestic ferret oocytes are epigenetically compatible with Black-footed ferret genomes. This was the first promising step in validating the use of interspecies cloning for genetic rescue.
A ROUTE TO INHERITABLE IMMUNITY IS CHOSEN
Revive & Restore also developed a research pathway to test if upregulating antibody expression for lifelong plague immunity offers a solution for inheritable immunity to the disease. Revive & Restore contracted the Texas A&M Institute for Genomic Medicine to develop a line of transgenic mice carrying an anti-plague antibody gene for eventual plague challenges at the US Geological Survey (USGS) National Wildlife Health Center.
REMOVING CANINE DISTEMPER BEGINS
ImQuest Biosciences began establishing a cell line experimentally infected with canine distemper virus (CDV) to test methods for eliminating the infection in culture. Once an effective method for clearing CDV infection is developed, the team will move on to removing CD-V from the historic cell line of the Black-footed ferret recorded as Studbook Number 2 (SB 2), thus preparing the SB2 cell line for cloning.
Revive & Restore received an Endangered Species Recovery Permit from the United States Fish & Wildlife Service after our application passed public review in compliance with the National Environmental Policy Act (NEPA). This first-of-its-kind authorization permitted the laboratory work necessary to demonstrate that the genetic rescue of Black-footed ferrets is feasible. Specifically, the permit allowed Revive & Restore and our partners to conduct two principal activities. First, determine the potential for using iSCNT cloning techniques to bring genetic diversity from historic cell lines back into the population. Second, it permitted Revive & Restore and our partners to test a variety of hypothetical sylvatic plague resistance solutions in cell culture.
Revive & Restore applied for a Recovery Permit with USFWS through the National Environmental Policies Act (NEPA) process. The application sought permission to conduct the laboratory-based experiments outlined in the 2016 proposal and was open to public comment through the Federal Register.
At an April 2016 meeting co-organized with San Diego Zoo Wildlife Alliance, a plan was set to facilitate recovery efforts of the Black-footed ferret through cloning and genetic engineering. The resulting proposal was then submitted to the Genomics Working Group. It outlined the initial laboratory steps necessary to address the genetic rescue of the Black-footed ferret on both fronts: optimizing the reproductive technologies necessary to increase genetic diversity and developing inheritable resistance to sylvatic plague. This draft proposal became the foundation for formal permit applications to the USFWS.
“A Road Map for 21st Century Genetic Restoration: Gene Pool Enrichment Of The Black-Footed Ferret” was published in The Journal of Heredity. The lead author was Dr. Samantha Wisely and co-authors included numerous members of the Genomics Working Group. The article charted out how to increase diversity for the Black-footed ferret using cloning, specifically the interspecies somatic cell nuclear transfer (iSCNT) technique.
Revive & Restore and the Black-footed Ferret Implementation Team (BFFRIT) met in person for the first time at the National Black-footed Ferret Conservation Center (NBFFCC). During the meeting, they formed the Genomics Working Group, which has continued to set annual meetings to coordinate the genetic rescue and genomic management of the Black-footed ferret. In partnership with the San Diego Zoo Wildlife Alliance Frozen Zoo® and Cofactor Genomics Revive & Restore initiated the building of a Black-footed ferret genomic database. We started by sequencing the genomes of Willa, SB2, Cheerio, and Balboa to measure the amount of genetic diversity we could expect to gain by genetic rescue efforts.
A PARTNERSHIP FOR GENETIC RESCUE BEGINS
After seeing our TEDxDeExtinction event online, Seth Willey from the United States Fish & Wildlife Service (USFWS) invited Revive & Restore to explore the potential use of genomic technologies to increase Black-footed ferret genetic diversity. The potential to clone new founders and use gene-editing technologies to develop plague resistance were discussed in those earliest meetings.