Cloning of Extinct and Endangered AnimalsKELLIE RABLEY12/4/2014

AgendaExample 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 pregnanciesoOnly 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

Results• One successful birtho Lived for at least 7 months

• Confirmed mitochondrial DNA was from the oocyte donor oUsed PCR amplification of 9 microsatellites

First birth of an extinct animal• Study revolves around the BucardooWent extinct in 2000

• Used fibroblasts derived from skin biopsiesoCame from a cryopreserved specimen

• Resulted in the birth of a female BucardooDied minutes within birth

Method• Embryos were reconstructed by fusing epithelial Bucardo cells and enucleated oocytes of domestic goatsoOocytes were from superovulated goats

• Two experiments were runoExperiment 1: Transferred at 3- to 6-cell stage then at the compact blastocyst stageoExperiment 2: Only transferred at 3- to 6-cell stage

• Embryos were either transferred into a Spanish ibex or hybridoHybrids were a crossbreed of Spanish ibex and domestic goat

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 cellsoUsed nine microsatellite markers

Future Projects and Outlook• Mexican gray wolfoDeclared extinct in MexicooReintroduced in New Mexico and Arizona

• Gastric brooding frogo Able to turn its stomach into a womboCryopreserved specimen is available

•Wooly MammothoSuccessfully sequenced its genomeoStill many challenges ahead

EthicsFOR

• Research Opportunitieso Life cycle of the organism o New treatments and antibiotics

•Conservation of species

AGAINST

• Animal Welfareo Loss of original habitato Birth defects

• Ecological Effects

• Cost

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.