Cloning of Extinct and Endangered Animals Kellie Rabley 12/4/2014

Agenda Example 1: Genetic rescue of a Mouflon Example 2: First birth of and extinct animal Future Projects and Outlook Ethics

Genetic rescue of the Mouflon The mouflon is an endangered wild sheep species Used cross-species nuclear transfer with post-mortem somatic cells Resulted in two pregnancies Only one successful birth

Method Collected cumulus-oocyte complexes (COC) from two Mouflon found in a field Dissociated granulosa cells from the COC and injected them into enucleated oocytes Activated with lonomycin and cycloheximide cultured in synthetic oviduct fluid The embryos then developed into blastocysts and were implanted into domestic females COC is mass of follicular cells that are surrounded by layers of cumulus cells, which helps protect the oocyte, which is the female gametocyte Everything was done in vitro with oocytes collected from Ovis aries, which is a closely related sheep species Blastocysts eventually become the embryo Total of 8 blastocysts developed, 4 were implanted domestic ewes, two became pregnant, one successful birth

Results One successful birth Lived for at least 7 months Confirmed mitochondrial DNA was from the oocyte donor Used PCR amplification of 9 microsatellites The lanes going from left to right are: Mouflon donor nuclei Mouflon clone Foster mother Oocyte donor

First birth of an extinct animal Study revolves around the Bucardo Went extinct in 2000 Used fibroblasts derived from skin biopsies Came from a cryopreserved specimen Resulted in the birth of a female Bucardo Died minutes within birth Last known Bucardo had tissue samples and frozen a year before it died Fibroblasts basically provide the structural framework for animal tissues

Method Embryos were reconstructed by fusing epithelial Bucardo cells and enucleated oocytes of domestic goats Oocytes were from superovulated goats Two experiments were run Experiment 1: Transferred at 3- to 6-cell stage then at the compact blastocyst stage Experiment 2: Only transferred at 3- to 6-cell stage Embryos were either transferred into a Spanish ibex or hybrid Hybrids were a crossbreed of Spanish ibex and domestic goat The thawed fibroblasts were derived from skin biopsies They superovulated the sheep to promote egg yield After experiment 1, no pregnancies came from the embryos implanted during the compact blastyocyst stage Couldn’t use domestic goats due to gestation period Used electrofusion to fuse the Bucardo cells and enucleated oocytes

Results One successful pregnancy out of 194 transferred embryos Newborn Bucardo died within minutes after birth due to deformed lungs Confirmed that the clone was genetically identical to the donor cells Used nine microsatellite markers Experiment 1: 30 transferred embryos into 5 recipients Two pregnancies, none were full term Experiment 2: 154 transferred embryos into 44 recipients Five pregnancies, only one carried full term Figure Values represent fragment sizes for both alleles from each microsatellite locus Left to Right: Newborn Bucardo Somatic nuclear donor cells from adult Bucardo female Surrogate hybrid Oocyte donor from domestic goat Control domestic goal

Future Projects and Outlook Mexican gray wolf Declared extinct in Mexico Reintroduced in New Mexico and Arizona Gastric brooding frog Able to turn its stomach into a womb Cryopreserved specimen is available Wooly Mammoth Successfully sequenced its genome Still many challenges ahead Mexican gray wolf Good example of the potential conservation of species Scientists were able to use the captive population to save them from complete extinction Gray wolves have been cloned with success using domestic dog oocytes and embryo recipients Even the generation gray wolf can be used as an oocyte donor and embryo recipient Gastric brooding frog These frogs went extinct in the 1983 Gave birth through their mouth Goal is to use study their ability to turn their stomach into a womb to create therapies and treatments for women who have trouble carrying their pregnancy to term Were able to successfully get a living embryo in 2013 Wooly Mammoth Genome was sequenced from a specimen found in Siberia Only 80% of the DNA corresponds to the mammoth due to degradation Will be difficult to obtain oocytes from an Asian elephant due to the anatomy of its reproduction system There is also a small amount of Asian elephants capable of breeding, along with the gestation period being around 2 years Currently the only successful technique would be artificial insemination

Ethics For Against Research Opportunities Conservation of species Life cycle of the organism New treatments and antibiotics Conservation of species Animal Welfare Loss of original habitat Birth defects Ecological Effects Cost Where will they live? Most extinct animals no longer have their original habitat. While we are able to clone animals, there is a small probability of obtaining a living organism. Let alone one without defects What if we were to introduce these into the wild? The natural cycle of an ecosystem would be disrupted by the new animal. Overall cloning methods are fairly expensive by itself. Once there is a clone, it also will need to be cared for properly (building a habitat, paying experts to care for it, other basic care fees) High cost, low yield

Questions or comments?

Resources Folch, J., M.j. Cocero, P. Chesné, J.l. Alabart, V. Domínguez, Y. Cognié, A. Roche, A. Fernández- Árias, J.i. Martí, P. Sánchez, E. Echegoyen, J.f. Beckers, A. Sánchez Bonastre, and X. Vignon. "First Birth of an Animal from an Extinct Subspecies (Capra Pyrenaica Pyrenaica) by Cloning." Theriogenology 71.6 (2009): 1026-034. Web. 23 Nov. 2014. Holt, W. V. "Wildlife Conservation and Reproductive Cloning." Reproduction 127.3 (2004): 317- 24. Web. 23 Nov. 2014. Loi, Pasqualino, et al. "Genetic Rescue Of An Endangered Mammal By Cross-Species Nuclear Transfer Using Post-Mortem Somatic Cells." Nature Biotechnology 19.10 (2001): 962. Food Science Source. Web. 1 Dec. 2014.