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Revive & Restore Scientific Publications

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November 13, 2018

Ben J. Novak

Abstract: De-extinction projects for species such as the woolly mammoth and passenger pigeon have greatly stimulated public and scientific interest, producing a large body of literature and much debate. To date, there has been little consistency in descriptions of de-extinction technologies and purposes. In 2016, a special committee of the International Union for the Conservation of Nature (IUCN) published a set of guidelines for de-extinction practice, Read More

establishing the first detailed description of de-extinction; yet incoherencies in published literature persist. There are even several problems with the IUCN definition. Here I present a comprehensive definition of de-extinction practice and rationale that expounds and reconciles the biological and ecological inconsistencies in the IUCN definition. This new definition brings together the practices of reintroduction and ecological replacement with de-extinction efforts that employ breeding strategies to recover unique extinct phenotypes into a single “de-extinction” discipline. An accurate understanding of de-extinction and biotechnology segregates the restoration of certain species into a new classification of endangerment, removing them from the purview of de-extinction and into the arena of species’ recovery. I term these species as “evolutionarily torpid species”; a term to apply to species falsely considered extinct, which in fact persist in the form of cryopreserved tissues and cultured cells. For the first time in published literature, all currently active de-extinction breeding programs are reviewed and their progress presented. Lastly, I review and scrutinize various topics pertaining to de-extinction in light of the growing body of peer-reviewed literature published since de-extinction breeding programs gained public attention in 2013.

Tom Maloney, Ryan Phelan, and Naira Simmons

Abstract: Horseshoe crabs have been integral to the safe production of vaccines and injectable medications for the past 40 years. The bleeding of live horseshoe crabs, a process that leaves thousands dead annually, is an ecologically unsustainable practice for all four species of horseshoe crab and the shorebirds that rely on their eggs as a primary food source during spring migration. Populations of both horseshoe crabs and shorebirds are in decline. Read More

This study confirms the efficacy of recombinant Factor C (rFC), a synthetic alternative that eliminates the need for animal products in endotoxin detection. Furthermore, our findings confirm that the biomedical industry can achieve a 90% reduction in the use of reagents derived from horseshoe crabs by using the synthetic alternative for the testing of water and other common materials used in the manufacturing process. This represents an extraordinary opportunity for the biomedical and pharmaceutical industries to significantly contribute to the conservation of horseshoe crabs and the birds that depend on them.

CRISPR Journal

Advancing a New Toolkit for Conservation: From Science to Policy

February 2018
Vol. 1, Issue 1, pp. 11-15

Ben J. Novak, Tom Maloney, and Ryan Phelan

Abstract: Climate change and non-native wildlife diseases are exacerbating persistent challenges to biodiversity such as habitat destruction, invasive species and over-harvesting. With these increasing threats there is a pressing need to expand the conservationists’ toolbox. Read More

CRISPR-Cas9 genome editing (GE) offers a precise and potentially transformative tool to confront these challenges. Researchers, regulators, conservationists and the public are all needed to engage proactively in the thoughtful and responsible development and application of these new tools.


Novak, Ben. “De-Extinction.” Genes. November 13, 2018.
Maloney, Tom; Phelan, Ryan; and Simmons, Naira. “Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection.” PLOS Biology. October 12, 2018.
Novak, Ben; Maloney, Tom; and Phelan, Ryan. “Advancing a New Toolkit for Conservation: From Science to Policy.” CRISPR Journal. February 2018: Vol. 1, No. 1, 11-15.
Novak, Ben; Estes, James; Shaw, Holland; Novak, Erika V; and Shapiro, Beth. “Experimental Investigation of the Dietary Ecology of the Extinct Passenger Pigeon, Ectopistes migratorius.” Frontiers in Ecology and Evolution. March 5, 2018: Vol. 6.


Novak, Ben et. al, “Natural selection shaped the rise and fall of passenger pigeon genomic diversity.” Science. 17 Nov 2017: Vol. 358, No. 6365, 951-954. Read More

Abstract: The extinct passenger pigeon was once the most abundant bird in North America, and possibly the world. Although theory predicts that large populations will be more genetically diverse, passenger pigeon genetic diversity was surprisingly low. To investigate this disconnect, we analyzed 41 mitochondrial and 4 nuclear genomes from passenger pigeons and 2 genomes from band-tailed pigeons, which are passenger pigeons’ closest living relatives.

Passenger pigeons’ large population size appears to have allowed for faster adaptive evolution and removal of harmful mutations, driving a huge loss in their neutral genetic diversity. These results demonstrate the effect that selection can have on a vertebrate genome and contradict results that suggested that population instability contributed to this species’s surprisingly rapid extinction.

Oehler, David A; Novak, Ben et. al, “Husbandry Protocols for the Band-tailed pigeon, Patagioenas fasciata albilinea, at the WCS, Bronx Zoo for future conservation management programs.” Zoo Biology. 15 Dec 2017: 1-8. Read More

Abstract: From 2015 to 2016 we determined the husbandry protocols involved in the captive rearing of the Band-tailed Pigeon (BTPI), Patagioenas fascinate albilinea, for use as a tool in the future management of like extant and extinct avian taxa. Current and historical ex-situ conservation management of BTPIs and the closely related Passenger Pigeon, Ectopistes migratorius, is limited in scope and required further examination. Focus on the BTPI within zoos and private aviculture facilities is currently lacking. New pressures on the wild populations and future examination of the parameters involved in the possible restoration of the Passenger Pigeon may rely on a complete understanding of these conservation management techniques. Here we report on the establishment of a colony of BTPIs, at the Wildlife Conservation Society (WCS), and detail the progress attained. A confiscated group of BTPIs was presented to WCS and allowed us to set up the colony, document the husbandry involved, and monitor neonatal development and the factors that influence that development. The information has provided a better understanding of the BTPI and has implications for the future conservation management of this and like species.

Kjelland, Michael E; Novak, Ben et. al, “Manipulating the avian egg: applications for embryo transfer, transgenics, and cloning.” Avian Biology Research. 3 January 2017: Vol. 10, No. 3, 146-155. Read More

Abstract: In vitro production of germline chimeras and avian cloning may utilise the transfer of avian embryos from their original eggshell to a surrogate eggshell for culture during incubation. Such embryo transfer is valuable for avian cloning as the only alternative would be to transfer the cloned avian embryos into the infundibulum of recipient birds. Given the advances in paleogenomics, synthetic biology, and gene editing, a similar approach might be used to generate extinct species, i.e. de-extinction. One objective of the present research was to examine if ratite eggs could be manipulated via windowing and sham injection, similar to that which could allow for avian genome manipulation and subsequent development. The efficiency of interspecific avian embryo transfer using Chicken (Gallus gallus domesticus) donor eggs and Turkey (Meleagris gallopavo) recipient eggshells was also investigated. Egg windowing and embryo transfer techniques utilised in the present research were adapted from those found in the scientific literature. Presumed fertile eggs from Rhode Island Red (n = 40), Silkie (n = 2), and White Leghorn Chickens (n = 18), Turkey (n = 48), Emu (Dromaius novaehollandiae) (n = 79), and Ostrich (Struthio camelus) (n = 89) were used in this research. Of the 41 Chicken eggs used for transfers into recipient Turkey eggshells, only one (2.4%) produced a chick. Of 31 windowed Emu eggs, one embryo survived for 25 d but no chicks were produced. Of 36 windowed Ostrich eggs, one embryo survived and hatched. The efficiency of the windowing and embryo transfers to produce chicks was low and further refinements are needed. Importantly, the results herein establish that manipulating ratite embryos is possible.


Piaggio, Antoinette J; Seddon, Philip J; Redford, Kent H; Phelan, Ryan et. al, “Is It Time for Synthetic Biodiversity Conservation?” Trends in Ecology & Evolution. 19 Nov 2016: Vol. 32, No. 2, 97 – 107.Read More

Abstract: Evidence indicates that, despite some critical successes, current conservation approaches are not slowing the overall rate of biodiversity loss. The field of synthetic biology, which is capable of altering natural genomes with extremely precise editing, might offer the potential to resolve some intractable conservation problems (e.g., invasive species or pathogens). However, it is our opinion that there has been insufficient engagement by the conservation community with practitioners of synthetic biology. We contend that rapid, large-scale engagement of these two communities is urgently needed to avoid unintended and deleterious ecological consequences. To this point we describe case studies where synthetic biology is currently being applied to conservation, and we highlight the benefits to conservation biologists from engaging with this emerging technology.

Soares, André; Novak, Ben J; Church, George M; Shapiro, Beth et. al, “Complete mitochondrial genomes of living and extinct pigeons revise the timing of the columbiform radiation.” BMC Evolutionary Biology 16 Oct 2016: Vol. 16, No. 1, 230.Read More

Abstract: Pigeons and doves (Columbiformes) are one of the oldest and most diverse extant lineages of birds. However, the nature and timing of the group’s evolutionary radiation remains poorly resolved, despite recent advances in DNA sequencing and assembly and the growing database of pigeon mitochondrial genomes. One challenge has been to generate comparative data from the large number of extinct pigeon lineages, some of which are morphologically unique and therefore difficult to place in a phylogenetic context.

Results: We used ancient DNA and next generation sequencing approaches to assemble complete mitochondrial genomes for eleven pigeons, including the extinct Ryukyu wood pigeon (Columba jouyi), the thick-billed ground dove (Alopecoenas salamonis), the spotted green pigeon (Caloenas maculata), the Rodrigues solitaire (Pezophaps solitaria), and the dodo (Raphus cucullatus). We used a Bayesian approach to infer the evolutionary relationships among 24 species of living and extinct pigeons and doves.


Wisely, Samantha M; Ryder, Oliver A; Novak, Ben J et. al, “A Road Map for 21st Century Genetic Restoration: Gene Pool Enrichment of the Black-Footed Ferret.” Journal of Heredity. Sept-Oct 2015; Volume 106, Issue 5, 581- 592.Read More

Abstract: Interspecies somatic cell nuclear transfer (iSCNT) could benefit recovery programs of critically endangered species but must be weighed with the risks of failure. To weigh the risks and benefits, a decision-making process that evaluates progress is needed. Experiments that evaluate the efficiency and efficacy of blastocyst, fetal, and post-parturition development are necessary to determine the success or failure or species-specific iSCNT programs. Here, we use the black-footed ferret (Mustela nigripes) as a case study for evaluating this emerging biomedical technology as a tool for genetic restoration. The black-footed ferret has depleted genetic variation yet genome resource banks contain genetic material of individuals not currently represented in the extant lineage. Thus, genetic restoration of the species is in theory possible and could help reduce the persistent erosion of genetic diversity from drift. Extensive genetic, genomic, and reproductive science tools have previously been developed in black-footed ferrets and would aid in the process of developing an iSCNT protocol for this species. Nonetheless, developing reproductive cloning will require years of experiments and a coordinated effort among recovery partners. The information gained from a well-planned research effort with the goal of genetic restoration via reproductive cloning could establish a 21st century model for evaluating and implementing conservation breeding that would be applicable to other genetically impoverished species.