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

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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. Questions or comments?

12. 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): Web. 23 Nov
Holt, W. V. "Wildlife Conservation and Reproductive Cloning." Reproduction 127.3 (2004): Web. 23 Nov
Loi, Pasqualino, et al. "Genetic Rescue Of An Endangered Mammal By Cross-Species Nuclear Transfer Using Post-Mortem Somatic Cells." Nature Biotechnology (2001): 962. Food Science Source. Web. 1 Dec