Proposal #1 (version 1.5) January 4, 2016

A proposal to generate new “founder” black-footed ferrets

via interspecies somatic cell nuclear transfer (iSCNT)

Submitted to US Fish & Wildlife Service,

National Black-footed Ferret Conservation Center (NBFFCC) through

The Genomics Working Group of the Species Survival Plan (SSP®) Subcommittee

of the Black-footed Ferret Recovery Implementation Team (BFFRIT)

For review in advance of the January 12, 2016 meeting

Submitted by Revive & Restore in partnership with San Diego Zoo Global

Contributors/ Editors/ Reviewers: Ben Novak, Science Coordinator, Revive & Restore, San Francisco, CA Oliver Ryder, Director of Genetics, San Diego Zoo Institute for Conservation Research Ryan Phelan, Executive Director, Revive & Restore, San Francisco, CA Martha Gómez, Independent consultant, Kodagen, Inc., Alabama; and the Veterinarian Sciences Doctoral Program, University of Conception, Chili John Engelhardt, Department of Anatomy and Cell Biology, University of Iowa, Iowa Michael Kjelland, Conservation, Genetics & Biotech, LLC, Vicksburg, MS Colleen Lynch, Animal Care Department, Riverbanks Zoo and Garden, Columbia, SC, and Conservation and Science Department, Lincoln Park Zoo, Chicago, IL Alejandro Camacho, Center for Land, Environment, and Natural Resources, University of California Irvine School of Law, Irvine, CA

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Table of Contents Introduction ............................................................................................................................. 3 Section 1. ................................................................................................................................ 4 Evaluation of advanced reproductive technologies and their application to black-footed ferret (BFF) genetic rescue Section 2. ................................................................................................................................ 8 Proposal to use iSCNT to increase gene diversity of the BFF breeding program via the generation of new Founders from San Diego Frozen Zoo Cell lines 2.1 The value of iSCNT to Black-footed ferret recovery ........................................... 8

2.2 iSCNT and the likelihood of success ................................................................... 8

2.3 Methods to produce BFF iSCNT clones ............................................................... 10

2.4 Assessing fitness of BFF iSCNT clones and clonal lineages ............................... 13

2.5 Special topics for experimental breeding and regulatory considerations ............. 13

Acknowledgments ................................................................................................................... 15 References ............................................................................................................................... 15

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Introduction

Over the course of three years, discussions between the US Fish &Wildlife Service Black-footed Ferret Implementation Team (BFFRIT), the Long Now Foundation’s Revive & Restore, San Diego Zoo Global, and members of the Black-footed ferret Genomics Working Group have resulted in a consensus to outline a proposal for the use of interspecies somatic cellular nuclear transfer (iSCNT) to further efforts to augment genetic diversity of the black-footed ferret (BFF), Mustela nigripes, breeding population. This proposal outlines the purpose, significance, methods, and recommendations for conducting research to produce the first BFF born from iSCNT. The proposed research goal is to establish yet another means of maintaining the genetic diversity of the BFF population in order to meet the long term goals set forth by the Species Survival Plan® (SSP).

The research proposed in this document, if deemed appropriate, will use BFF cell cultures, which are the property of the United States Fish and Wildlife Service, requiring permission to access and handle. This work will also require extensive laboratory resources and personnel, necessitating substantial funds, which Revive & Restore and project partners intend to secure independent of USFWS funds. The ultimate integration of cloned ferrets and their progeny into the captive breeding population and eventual release to the wild requires attention to regulatory barriers, as such integration of cloned individuals is unprecedented. To date, no wild animal clone or progeny of a clonal lineage has ever been released to the wild despite the production of healthy clones1 and clonal lineages2. Fundraising for such a project would be difficult without the support of the BFF SSP stakeholders. Undertaking such an endeavor will require the ability to navigate USFWS regulatory frameworks to achieve the use of BFF clones for genetic rescue. Given these parameters it is important to reach affirmative consensus agreements before fundraising and conducting research begins so that the products of the proposed research do not result in wasted efforts. Therefore, our aim in this proposal is threefold:

1. We request consent from the BFF SSP stakeholders to reference their affiliated support for

the research described in detail below when seeking funds to conduct the proposed research.

2. We seek to permission to obtain and handle the necessary biological resources to carry out the research proposed among the various collaborating institutions that will partner in this venture (e.g. BFF fibroblast cell cultures, semen samples, etc).

3. We want to establish a commitment from the BFF SSP to facilitate the regulatory approval process to integrate cloned ferrets and their progeny into the captive breeding program, conditional, of course, on the results of risk assessment/evaluation as per the agreed protocols of the BFF genomic committee.

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Section 1. Evaluation of advanced reproduction technologies and their application to BFF genetic rescue

Many genomic and advanced reproductive technologies have been developed for model

organisms, several of which have been applied to wildlife. Pedigree analytics in particular has been aided by genetic studies to evaluate the genetic management of breeding programs and to alleviate inbreeding depression3. Artificial insemination (AI), in vitro fertilization (IVF), interspecies embryo transfer of cryopreserved embryos4,5, and interspecies somatic cell nuclear transfer (iSCNT)6 have been performed with success on various wildlife species. More recent projects have produced induced pluripotent stem cells (iPSCs) of endangered species7. Such cells could someday be used for IVF or even parthenogenic embryogenesis; iPSCs have been reprogrammed into germ-cell like cells that produced live mouse pups8, but this technology is not yet proven beyond rodent models, and therefore is not applicable at present for BFF genetic rescue efforts.

Here we review some reproductive technologies (table 1) and present a comparison of

their relative value to BFF recovery (table 2). Such technologies, as discussed in previous meetings, can facilitate maintaining the BFF population’s calculated gene diversity (GD) more efficiently, augment existing GD, and open the door for more advanced forms of conservation such as transgenic facilitated adaptation to confront one of the BFF’s greatest conservation challenges, exotic disease.

It should be evident when evaluating potential technologies that any technology that

requires the sacrifice of black-footed ferrets is sub-optimal. Current methods of oocyte collection are conducted post-mortem, more for economic convenience than for technical reasons. It may be simple to optimize laparoscopic procedures for oocyte collection following superovulation, which has been done extensively in felids. Some 1,603 live laparoscopic oocyte retrievals have been conducted on 337 domestic cats over 15 years of Martha Gómez’s career (personal communication). Repeated oocyte retrieval did not affect oocyte quality for the production of iSCNT felid embryos. It should not be difficult to conduct similar procedures for BFF oocyte retrieval. However, for cloning purposes, the number of oocyte retrievals required for successful production of clones would involve the superovulation of as many as 20-40 jills for each cloning attempt. This would remove those jills from use during natural breeding cycles. The low efficiency of live offspring means that a large number of oocytes that could potentially become viable offspring would be lost. The collection of oocytes for IVF is a far more efficient use of BFF oocytes for the production of offspring. Though iSCNT may have lower efficiency rates than that of intra-species SCNT, we propose that to maximize genetic rescue gains while protecting BFF biological resources the domestic ferret, Mustela putoria furo, should be used as the oocyte donor and surrogate mother for iSCNT.

Cryogenic preservation of embryos offers a very attractive means of managing the

reintegration of genetic diversity of individuals over the course of lengthening generation gaps--such as embryos derived from current successful genetic rescue efforts derived from crossing historic sires with living dams via AI9. Cryopreserved embryos could be transferred into BFF females in which matings fail to conceive (sterile matings in domestic ferrets produce pseudo-pregnant females capable of successful embryo implantation for surrogacy). A mated BFF in which conception failed may also be viable for embryo implantation, though detection of failed

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pregnancy may not be possible in the window of time necessary to transfer an embryo successfully. Alternatively, due to the low number of female BFFs, domestic ferrets can be used as surrogate mothers to carry pregnancy of BFF cryopreserved embryos, allowing for more offspring to be produced while BFF mothers foster naturally or artificially inseminated pregnancies. Embryos for cryopreservation and future transfer could be collected surgically from pregnant dams when offspring could be considered surplus or created using iSCNT and IVF.

Artificial insemination procedures have already been established and used for genetic

rescue of BFF9. This technology is relatively inexpensive with moderate efficiency; consequently the use of this technology is rated highly in the scoring. However, cryopreserved spermatozoa for the use of genetic rescue suffers disadvantages: 1) most samples do not possess the ability to introduce new founders to the breeding program; 2) samples from the founder generation are a limited resource; 3) motility in older BFF spermatozoa was found to be lower than samples from more recent generations; and 4) artificial insemination presents a male bias for genetic rescue; no valuable female lineages are retrievable via this method. Unless IVF is implemented with cryopreserved oocytes, spermatozoa from any generation in the BFF program must always be mated with living females, reducing the full potential for rescuing rare alleles, increasing GD, and lowering inbreeding coefficients.

iSCNT offers a means to generate valuable individuals, representing high theoretical

values of GD of either sex, as long as cryopreserved or frozen material exists with intact nuclei. Two source populations of such material exist10, one of which consists of cryopreserved fibroblasts of non-founder ferrets that were captured alongside the breeding program founders at Meeteetse, WY. These are the cell lines that we propose using for initial iSCNT cloning trials. The second source are tissues preserved in a standard freezer (-20C) from Mellette County, South Dakota.

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Table 1 Genomic and advanced-reproduction technologies in regards to BFF genetic rescue.

Technology Proven model system Important Considerations

Application to BFF recovery Mammal species

Domestic ferret BFF Efficiency a Innovations for use with BFF Caveats

Artificial insemination (AI) b ✕ ✕ ✕ 30% none necessary limited founder samples maintain/reintegrate valuable male

lineages In vitro fertilization (IVF)c ✕ 35% non-lethal oocyte collection diverted bioresourcesg maintain/reintegrate valuable

female lineages Cryopreserved embryo transfer d ✕ ≤20% establish IVF/embryo

collection diverted bioresourcesg preserve & reintegrate valuable individuals

iSCNTe ✕ ✕ ≤5% none necessary animal welfare costsh generate new founders iPSC facilitated embryogenesisf ✕ %22 iPSC & gametogenesis

research extensive research needed Alternative to iSCNT from fibroblasts

a) All efficiencies are derived final stage efficiencies not factoring the efficacy of preceding steps, except for AI. b) Efficiency represents average percentage of viable sperm capable of fertilization10. c) The rate of successful fertilization in vitro is 30-75% among in mice and humans. An average of 35% of implanted embryos result in live births.11 d) The rate of cryopreserved embryos forming blastocysts for implantation was 22-26% in African wild cats.5 e) Prior to producing live ferret offspring many steps apply with varying efficiency rates: oocyte-donor cell fusion, in vitro cloned embryo incubation, and implantation to

surrogate mothers. With domestic ferrets live births occurred for 1-6% of implanted cloned embryos after a “donor cell rejuvenation step”, by using donor cells from 21 day old fetuses cloned from the original senescent donor cells. The efficiency of producing the first clones is 0.8-2%.12

f) 67% of iPSC derived oocytes (termed primordial germ cell like-cells) successfully fertilized via IVF and 69% of those fertile embryos developed to the blastocyst stage for implantation in mice.8

g) The use of jills for oocyte collection versus breeding to raise litters can be seen as a diversion of resources to less valuable recovery means--though this is debatable. h) While the use of domestic ferrets as surrogates oocyte donors and mothers eliminates diversion of BFF bioresources, some pregnancies will fail and some pups will die

prematurely, which involves the loss of life and likely the discomfort and suffering of both surrogates and offspring.

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Table 2 Scored comparison of genomic and advanced reproductive technologies for BFF recovery.

Technology Proven model

Developments needed for BFF Efficiency Negative BFF

impacts BFF value Conservation value to other species Total score

AI 1 1 1 1 0 0 4 IVF 1 0 1 0 0 0 2 Cryopreserved embryo transfer 1 0 0 0 1 0 2 iSCNT 1 1 -1 1 1 0 3 iPSC facilitated embryogenesis 1 -1 0 0 1 1 2

Table 2 Scoring Key

Scored parameter -1 0 1 Proven model No - Yes Developments needed for BFF None Moderate Extensive Efficiency Low Moderate High Negative impacts to BFF High Moderate Low BFF value cons outweigh pros Limited returns High potential Conservation value to other species - precedent unprecedented

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Section 2. Proposal to use iSCNT to increase gene diversity of the BFF breeding program via the generation of new founders from San Diego Zoo Global’s Frozen Zoo® (SDFZ) Cell lines Valuable information regarding the value of cloning BFF, as well as the need for applying cloning through a logic framework, is presented by Wisely, et al. in the white paper “A road map for 21st century genetic restoration: gene pool enrichment of the black-footed ferret” 13. In this proposal we will explore further considerations for BFF cloning prompted by the white paper and present a detailed methodology for producing BFF clones. 2.1 The value of iSCNT to Black-footed ferret recovery

The BFF recovery program has had a long history of careful and methodical management to maintain a viable breeding population, notably setting precedents for the use of advanced reproduction technologies for the purpose of genetic rescue9. For the exact same purposes that AI was used to produce offspring representing a gap of nearly 20 generations between sire and dam (restoring diversity from breeding population founders), iSCNT should also be pursued to increase genetic diversity (as measured by theoretical GD) – through the generation of new founders, which is unprecedented in previous genetic rescue efforts for this species.

At the SDFZ two cell lines are cryopreserved: a female BFF (SB10) and a male BFF (SB2), both captured at Meeteetse, WY, between 1985-1987. Neither of these individuals has living descendants in the BFF breeding program. Full genome sequencing and comparison carried out by Revive & Restore has shown that these cell lines contain notably higher counts of unique alleles and heterozygosity when compared to ferrets of the present generation (data available under NCBI Bioproject PRJNA254451). SB10 and SB2 clones would effectively integrate 2 new founders into the BFF breeding population pedigree, increasing the number of founders by nearly 30%, from 7 to 9 individuals. In small closed populations even the introduction of a single new founder has shown to have major benefits in reversing inbreeding depression14.

The female SB10, though less genetically diverse than the male SB2 (resulting from

whole genome comparisons, unpublished), represents potentially the only viable means to recover a female breeding line from the founding generation. Clones of SB10 could be artificially inseminated using the limited founder generation semen samples to produce offspring with significantly lower inbreeding coefficients than could otherwise be produced. A mate pairing between an SB10 and SB2 clones would introduce a new lineage to the existing BFF pedigree, producing the greatest benefit as measured by theoretical GD. Ultimately there are many potential permutations for breeding clones of SB10 and SB2 to maximize their genetic-rescue impact.

2.2 iSCNT and the likelihood of success Successful production of iSCNT clones of non-model organisms has had low rates of efficiency, though many combinations of donor nuclei and oocyte recipient have been attempted8. Three reasons have been proposed to explain the low efficiency of cloning:

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1. The recipient oocyte may not efficiently reprogram the methylation sites of the donor nucleus, a process necessary for the donor genome to differentiate the development of multiple tissue types. In lay terms, the genome of a skin cell is programmed to form skin, not whole organisms15. However, epigenetic anomalies of clones are generally thought to not persist in offspring.

2. A problem specific to iSCNT is the potential incompatibility between the encoded gene products of the oocyte’s mitochondrial DNA and that of the donor nuclear genome8. Since the two genomes are from different species they may not interact properly to carry out vital cell functions16.

3. Differences in pregnancy gestation (e.g. duration, hormone release, etc.) between the surrogate mother species and the cloned embryo species may inhibit embryonic development to parturition. Such was the case with the Bucardo when attempts were made to use domestic goats as surrogate mothers17.

Table 3 displays the genetic distances of mitochondrial genomes between iSCNT species

pairs (alignments of genomes performed in Geneious R818). Divergence times were derived from published literature using DNA molecular clocks alone as well as DNA molecular clock phylogenies calibrated with fossil data19-24. As can be seen, the most successful iSCNT projects consisted of species pairs with high mtDNA pairwise identity (>90%) and divergence times under 4 million years. The successful pairs were also of species similar in live birth mass and gestation period. Table 4 displays results of mtDNA/nuclear genome communication compatibility found by producing cybrid cell cultures. A cybrid is a cell in which the native mtDNA has been removed and replaced by that of another individual. In both humans and mice functional respiration was demonstrated between the components of the mtDNA and nuclear genomes between species pairs that diverged <10 million years ago.

Table 3 Comparison of proposed BFF/Domestic ferret interspecies mtDNA genetic distance to other iSCNT combinations6.

Nucleus Donor Oocyte Donor Evolutionary divergence (mya)

MtDNA Identity Pregnancy Live

Births Offspring survived

to adulthood Black-footed ferret Domestic ferret 0.60000000 98.0% - - - Zebu Domestic cow 1.00000000 98.5% Yes Yes Yes Banteng Domestic cow 4.00000000 99.9% Yes Yes Yes Guar Domestic cow 4.00000000 93.4% Yes Yes No* Giant eland Domestic cow 13.20000000 86.0% No - - Domestic sheep Domestic cow 16.20000000 84.7% Yes No - Domestic pig Domestic cow >50.00000000 80.0% No - - African wildcat Domestic cat 2.00000000 97.5% Yes Yes Yes Sandcat Domestic cat 3.20000000 96.8% Yes Yes No Black-footed cat Domestic cat 4.50000000 92.0% No - - Leopard cat Domestic cat 11.50000000 89.6% Yes No - Korean tiger Domestic cat 16.20000000 87.6% No - - Rabbit Domestic cat >75.00000000 75.0% Yes No - Wolf Domestic dog >0.02000000 99.8% Yes Yes Yes Coyote Domestic dog 2.50000000 95.5% Yes Yes Yes Mouflon Domestic sheep 0.38000000 99.8% Yes Yes No Argali Domestic sheep 3.21000000 97.3% Yes No - Bighorn sheep Domestic sheep 5.63000000 94.3% Yes No - Bucardo Domestic goat 2.20000000 96.8% Yes Yes No

*Guar died from dysentery unrelated to birth defects, therefore mtDNA incompatibility was not likely a problem.

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The BFF and domestic ferret, as can be seen in table 3, represent the closest related species pair (by divergence time) of any interspecies clone ever produced. Their mtDNA pairwise identity falls into the range of other successful iSCNT clones. Furthermore, the BFF and domestic ferret are similar in size and have identical gestation periods of 42 days25. Revive & Restore’s current whole genome comparisons reveal that the two species’ genomes are 99.6% identical. The majority of gene differences present are silent mutations not affecting phenotype. For these reasons we conjecture that iSCNT of the BFF using domestic ferret donor oocytes and surrogate mothers will be successful with similar efficiency rates of SCNT cloning of domestic ferrets12.

Table 4 Divergence times26-28 of successful and unsuccessful cybrids6.

mtDNA Donor Cell recipient

Evolutionary divergence (mya)

mtDNA-nuclear compatibility

Chimpanzee Human 6.600000000 Yes Bonobo Human 6.600000000 Yes Gorilla Human 8.300000000 Yes Orangutan Human 16.500000000 No African green monkey Human 31.600000000 No Squirrel monkey Human 43.500000000 No Lemur Human 87.200000000 No Japanese wild mouse Mouse >2.000000000 Yes Algerian mouse Mouse 2.000000000 Yes Ryukyu mouse Mouse 5.000000000 Yes Gairdner’s shrewmouse Mouse 7.000000000 Yes Rice-field mouse Mouse >8.000000000 Yes Vlei rat Mouse 10.000000000 No Brown rat Mouse 10.300000000 No Syrian golden hamster Mouse 24.700000000 No Chinese Hamster Mouse 24.700000000 No 2.3 Methods to produce BFF iSCNT clones

Given the assertions stated in section 2.2 we propose to clone BFF embryos with fibroblast donor cells by using the same protocols optimized for domestic ferret cloning presented by Sun et al12 with slight modifications for iSCNT specific problems, such as epigenetic reprogramming. Our proposed research is as follows:

1. The first donor cells selected for BFF iSCNT proof of concept we propose to be SB6815

for two reasons: a. The first cloned BFF will be used to optimize cloning protocols for use with the

historic cell lines. This will require multiple cloning attempts. Early passage donor fibroblasts yield higher success rates when cloning. The cell culture of an extant ferret is less of a limited resource than using cell lines of long-deceased individuals, as more fresh cells can be collected from the extant donor individual prior to or upon death (SB6815 is now an older ferret (5 years of age, per communication with Colleen Lynch), the time-window of collecting new tissue samples is likely soon to close; in the event that SB6815 dies before initiation of this work we would request that multiple tissue collections be conducted post-mortem to secure a large resource of cell cultures).

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b. SB6815 is an extant ferret of high GD value and has yet to produce offspring due to behavioral traits. Behavioral traits are unlikely to be replicated in clones. A clone of this male extends the “breeding life cycle” of this individual for more breeding attempts.

2. Twenty domestic ferret jills can be superovulated using a combination of 100. I.U. equine chorionic gonadotropin and 100 I.U. human gonadotropin29, however mating jills to vasectomized males yields oocytes of higher quality for cloning (unpublished, pers. communication with John Engelhardt). First collection attempts will be done with trial live laparoscopic procedures. If this procedure does not prove feasible in ferrets, the jills will be euthanized for standard post-mortem oocyte retrieval. The target number is 300 oocytes (expecting ~15 per female). Mature oocytes will be enucleated for cell fusion with BFF donor fibroblasts.

3. Early passage fibroblasts of SB6815 will be screened for abnormalities and viral infection. Healthy donor cells will be injected under the vitelline membrane and fused to the enucleated oocyte with an electrical shock – this causes the cell membranes of the donor cell to merge with the cell membrane of the oocyte, introducing the entire cytoplasmic contents of the donor fibroblast into the enucleated egg cell. This results in a reconstructed (cloned) single cell embryo that contains both domestic ferret mtDNA and BFF mtDNA. Cell fusion methods have shown to yield higher rates of efficiency in ferrets12, likely due to maintaining the integrity of the donor cell’s mtDNA/nuclear cellular pathways, though conflicting data exists concerning the positive, neutral, or negative impact of donor/recipient heteroplasmy in regards to the efficiency of producing healthy offspring6.

a. A subset of donor cells and or/cloned embryos can be chemically treated to alleviate epigenetic incompatibilities, elevating efficiency of oocyte reprogramming of the donor cell genome--as done in previous work with felids16. Efficiency of live births from treated and untreated donor cells will be taken into account.

4. Cloned embryos will be developed in vitro for 24 hours to the two-cell stage and then transferred into pseudo-pregnant jills (at least 50 reconstructed embryos should be transferred into at least 4 jills). Mating to vasectomized males induces Jill pseudo-pregnancy. Approximately 2-4 viable fetuses can be expected.

5. At this point two experiments will be performed: One set of established pregnancies will be allowed to come to term to produce live BFF iSCNT clones. In the event of complications, birth will be induced or caesarean section will be performed. From 200 transferred embryos we can now expect up to 1-2 live births.

a. A second set of established pregnancies will be terminated at 21 days of gestation to harvest fibroblast cells from the fetuses. At this point donor cell cultures will be examined for karyotype abnormalities and to verify the integration of donor cell genomes with PCRs or SNP arrays. Fetuses displaying proper genomic structure and exhibiting markers of good developmental health will be used for subsequent steps.

b. These fibroblasts are the “rejuvenated” donor cells that will be used to repeat steps 2-4. This time embryos will be brought to term and live births will occur naturally. In the event of complications, birth will be induced or caesarean section will be performed. From 300 transferred embryos we can now expect up to 6 live births using these rejuvenated cells if results from Sun et al. in domestic ferrets are replicated. These are the numbers expected in best-case scenario efficiency.

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Previous cloning work used embryonic fibroblasts, which have higher success rates in cloning – for work with adult somatic fibroblasts we should expect to require three times as many oocytes to result in 6 live births.

6. All live offspring will be screened genetically to verify that they are indeed clones of SB6815. The most efficient form of cloning--standard versus rejuvenated--will be used for the SB2 and SB10 cell lines.

The highly valuable SB2 cell line has tested positive for canine distemper virus (CDV)

cytopathology, present due to the ferret’s death from the disease in 1985. A clone from SB10 is likely to die during development unless CDV can be removed, which could be accomplished in a number of ways. CRISPR/Cas9 technology, which has been used to suppress and prevent pathological expression of HIV-1 provirus and Hepatitis B viruses in human cell cultures31-33

could be used to attempt removing CDV. CRISPR/Cas9 is an endonuclease used by bacteria to cleave viruses upon infection34; this bacterial immune function of CRISPR/Cas9 works just as efficiently in mammalian cells. In the referenced studies viral DNA was cleaved in the cytoplasm, nucleoplasm, and even in the host genome at integration sites without off-target damage to the host genome. A Cas9 variant extracted from the bacteria Francisella novicida has recently been shown to target and cleave viral RNA infecting eukaryotic cells35; the same Cas9 variant would be used to target and disintegrate the CDV RNA genome. Cas9 guide RNAs can be designed to cleave multiple genes of the CDV genome (GenBank accession NC_001921.1,36) but leave the BFF genome unharmed. Other methods to suppress negative effects of CDV in the SB2 cell lines would be the use of RNAi technology or CDV specific antibodies to neutralize the proliferation of the virus.

Cloning the female SB10 will be conducted following steps above with one alteration to step 5a in the event that rejuvenated donor cell cloning is chosen. To assess integration of donor DNA in step 5 of the first cloned fetuses, low coverage whole-genome shotgun sequencing will be used rather than PCR/SNP arrays. Justifying the added cost of this analysis, it will allow us to assess the relative copy number of BFF mtDNA genomes to domestic ferret mtDNA genomes. The reasoning for this is explained in section 2.5. Further cloning efforts for BFF should continue after successful clones of SB6815, SB2, and SB10 are produced. The source this time would be the second population of donor cell material: frozen tissues of Mellette County ferrets from the first BFF captive breeding efforts, which ultimately failed (1972-1979). While these tissues were not cryopreserved, and intact viable nuclei will be rare and difficult to obtain, these ferrets represent the only potential viable cellular material that meet the criteria of traditional genetic rescue: the introgression of alleles into an impoverished population by interbreeding individuals from a separate source population. The SDFZ cell lines of SB2 and SB10, while contributing new founders, are members of the same population that gave rise to all living BFF. From a genetic rescue standpoint, the Mellette county specimens are likely to introduce more unique alleles than SB2 and SB10 since they represent a completely separate population from the source of living BFF (Meeteetse, WY); whereas SB2 and SB10, though lacking living descendants, are still Meeteetse ferrets. Cloning in similar fashion to that described here can be conducted using cells from the Mellette County frozen tissues, but intensive search for intact nuclei must precede donor cell selection. Cloned pups of mice have been produced from tissues of the same condition37. Thoroughly comprehensive genomic studies of pre-bottleneck BFF populations should be analyzed before pursuing cloning of the Mellette County specimens, given the potential for outbreeding

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depression. With significant effort, clones of Mellette County ferrets can likely be generated, though this proposal is restricted to the immediate pursuit of cloning the SDFZ fibroblast cell lines. 2.4 Assessing fitness of BFF iSCNT clones and clonal lineages The fitness of iSCNT cloned individuals and their offspring may not be equivalent to ferrets born from normal fertilization. It is possible that the presence of domestic ferret mtDNA may affect cellular pathways in unpredictable ways. This is not entirely unprecedented, as hybrids between BFF and Siberian polecats exhibited aberrant behavior (personal communication, Dean Biggins & Paul Marini). There is a risk not only of reduced fitness due to the cloning process, but also that unique alleles present in SB10 or SB2 may actually be deleterious to such degrees that the desired benefits of genetic rescue are not observed in their progeny.

In order to assess the viability of using iSCNT clones, and specifically SB10 and SB2

clones, we propose developing a separate experimental breeding population using surplus BFF individuals from the captive breeding program. A facility will be constructed or designated in such a manner that there is no risk of interbreeding between the experimental clonal population with other captive bred or wild released ferrets should any clones or progeny escape.

The same measures used to assess fitness traits currently in BFF should be sufficient to assess the fitness of the experimental group. The number of generations as well as the independent permutations of the experimental pedigree animals should not only satisfy scientific analysis but also satisfy regulation and policy conditions. Therefore the appropriate pedigree design for the experimental population should incorporate current managers of the BFF pedigree, scientists experienced in analyzing BFF fitness, and the necessary representatives of agencies that may bare relation to the use of clones and their offspring for endangered species recovery. We hope that the conditions of such a design can be outlined by the BFF genomic advisory committee following the 2016 meeting, should this requested element of this proposal be granted (outlined in the introduction). The exact protocols of risk assessment for clones should not be considered pertinent to the initial decisions to support and pursue cloning experiments, given the amount of time that will be required to secure funds and produce successful clones.

2.5 Special topics for experimental breeding and regulatory considerations

The F1 generation iSCNT BFF clones can likely be expected to contain a mixture of domestic ferret and BFF ferret mtDNA genomes. This does not, however, classify them scientifically as hybrids. Hybrids are the result of interbreeding between two separate species, in which the result is a mixture of the parent species’ nuclear DNA but not a mixture of the parent species’ mtDNA. The genotype of the mtDNA is always inherited from the mother. In nature there are examples of species in which the majority of the nuclear genome is of one species, but the mtDNA is from another (e.g. Baranoff island grizzly bears38). The descendants of hybrids carry nuclear genome signatures of hybridization, even thousands of generations later (e.g. humans possess Neanderthal alleles from hybridization >30,000 years ago39). The exact classification of the iSCNT BFF ferrets, which possess BFF mtDNA haplotypes, is that of a heteroplasmic interspecies cybrid. To the knowledge of current biology, such creatures do not naturally occur (but they potentially could).

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In Wisely, et al13, a breeding regime is described to purge the domestic ferret mtDNA

from future generations descended from our F1 cybrids. When breeding male iSCNT clones, their progeny do not inherit the mtDNA, and therefore the domestic ferret mtDNA is immediately purged. When breeding iSCNT SB10 female clones, the proposed strategy is to selectively breed only the resulting male offspring of clones so that the domestic ferret mtDNA is purged by the second generation. This process eliminates the use of valuable female progeny, and may not be necessary.

Heteroplasmy is present in nearly all eukaryotic organisms to varying degrees. The D-

loop region of the mtDNA genome has hypervariable repeat regions that often mutate during DNA synthesis in cell division, generating multiple genotypes40,41. It was assumed that these multiple genotypes are inherited by daughter cells randomly, but analysis of the tissues of naturally occurring heteroplasmic mice revealed that selection favored one genotype over another based on the cellular needs of any given tissue42. Even more relevant to the iSCNT cloning of ferrets was the observation of differential donor cell to oocyte recipient species’ mtDNA haplotypes in different tissues among guar-cow iSCNT fetuses43. The percentage of gaur mtDNA in different fetus tissues ranged from 0.7-99.0%.

This presents the possibility that germ-lines of iSCNT female clones and their female

progeny may naturally purge domestic ferret mtDNA, especially given that cellular functions should favor communication between the BFF nuclear genome and BFF mtDNA genome over communication between BFF nuclear genome and domestic ferret DNA. This is the reasoning for sequencing low coverage genomes in step 5 for the iSCNT cloning process of SB2. (When analyzing copy number of mtDNA haplotypes in guar-cow iSCNT clones, qPCR was used; however the genetic similarity between BFF and domestic ferret haplotypes would make this technique potentially uninformative). By selecting donor cells with higher ratios of BFF mtDNA to domestic ferret DNA for the rejuvenated cloning steps we can preserve the presence of BFF mtDNA during the second round of cloning and may foster the natural purging of domestic mtDNA by giving the BFF mtDNA the advantage of numbers during random segregation. Before rejecting the use of female clonal progeny in continued breeding, the somatic cells and germ lines of every female offspring should likewise be screened for ratios of BFF mtDNA to domestic ferret mtDNA. In this way all valuable individuals can breed, and the purging of domestic ferret mtDNA can simultaneously be achieved. If the process does not work after several generations, then the integration of clonal lineages can revert to the male biased method13.

In the event that domestic ferret mtDNA cannot be fully purged from detectable clonal

offspring, we strongly urge that clonal progeny not be considered hybrids, though if they must, it should not stand as a significant issue to BFF recovery. The genetic rescue of the BFF should outweigh concerns of hybridization, especially given recent discoveries into the extensive hybridization of common and endangered species, such as wolves, bison, polar bears, etc. It should be considered that if the presence of domestic ferret mtDNA does not alter BFF traits or fitness, then preventing its admixture to the BFF population may be unnecessary and irrelevant from biological standpoint.

The generation of domestic ferret ♂/BFF ♀ hybrids as oocyte donors would circumvent

these issues regarding mismatching nuclear/mtDNA. While domestic ferret oocytes are likely

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compatible with BFF nuclear genomes, domestic ferret ♂/BFF ♀ hybrid females would provide oocytes that carry BFF mtDNA. The resulting iSCNT clones would carry BFF nuclear and BFF mtDNA and no domestic ferret DNA. This would reduce concern for incompatibilities in the cloned embryos, potentially increase the efficiency of cloned embryo development, and remove any issues of cybrid classification from a regulatory standpoint; all genotyping/breeding steps outlined previously concerning the purging of domestic ferret mtDNA would not be necessary.

Only a few female BFFs would be needed to generate several litters for breeding an

oocyte donor population, which can also act as the embryo recipient population (surrogate mothers). Hybrid surrogate mothers may yield higher implantation and pregnancy success rates due to the presence of BFF maternal alleles. Hybridization of BFFs for research purposes is not unprecedented44. BFFs produce fertile hybrids with Siberian polecats44 and should produce fertile hybrids with domestic ferrets, as domestic ferrets and Siberian polecats also produce fertile hybrids45. Acknowledgments The information presented in this proposal stems from research and consultation by Martha Gómez. We thank Pete Gober for the opportunity to submit this proposal for discussion at the 2016 Genomic Advisory Group meeting. This proposal would not be possible without the previous efforts of the white paper authors Samantha Wisely, John Engelhardt, and Oliver Ryder. Special thanks to Seth Willey for engaging Revive & Restore in BFF conservation. References

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