1. Reversing Species Extinction By Mammalian Cloning
Haley Kuhn
PBIO 4500
September 30th, 2017

2. The Loss of Biodiversity
801 extinct species
3,879 critically endangered species
5,689 endangered species
Click to add text
Graph adapted from IUCN data presented in Coballos et. al. 2015

3. Animal cloning can increase biodiversity
Interspecies Somatic Cell Nuclear Transfer (iSCNT) Cloning
Closely related domestic animal species
Example studies:
Domestic cat oocyte cytoplasm supports development of African wild cat (2004)
Domestic cattle oocyte cytoplasm supported proliferation of a gaur (2012)
Canine oocyte cytoplasm supports development of endangered wolves (2007)
Mouflon was produced using a goat as a surrogate mother (2001)

4. Cloning of the Bactrian Camel
Credit: Nisar Ahmad Wany, Binoy S. Vettical, Seung B. Hong
Reproductive Biotechnology Center, Dubai, UAE
Camelus bactrianus
Camelus dromedarius
8th most endangered mammal
Donated skin fibroblast cells
Was successfully cloned
Oocyte donor / surrogate mother

5. Preparation Animal preparation: Ovarian stimulation
Ionomycine/6-dimethylaminopurine (6-DMAP)
Follicle-stimulating hormone
Recipient cytoplast preparation: Enucleation
Selection of oocytes
Manipulation medium (Hepes-TCM-199)
UV light exposure
Donor karyoplast preparation: Obtaining nuclei
Ear skin biopsy
Fibroblast establishment
Enzymatic solution

6. Procedure Nuclear transfer, fusion, and activation
2 DC pulses of 100V for 15 μs each
Ionomycine/6-DMAP
Activated oocytes to embryo culture medium I 
Cleaved embryos to embryo culture medium II
Hoechest staining and cell counting
Embryo Transfer
Blastocyst stage
Bull parade pregnancy detection

7. Results Healthy calf was born after 392 days of gestation
Microsatelitte analysis
Died on day 7 to acute septicemia
Significantly higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with BT-SKF cells

8. Capra pyrenaica pyrenaica
Cloning of the Bucardo
Capra pyrenaica pyrenaica
Capra aegagrus hircus
1 of 4 subspecies of the Spanish ibex
Last bucardo died in 2000
Cells were obtained by skin biopsy, multiplied, and kept frozen in liquid nitrogen
First extinct subspecies to be successfully cloned

9. Preparation Animal preparation: Ovarian stimulation
Highly purified porcine FSH and LH
6 intramuscular doses of pFSH
Recipient cytoplast preparation: Enucleation
Selection of oocytes
Manipulation medium (M199-Hepes)
UV light exposure
Donor karyoplast preparation: Obtaining nuclei
Frozen skin biopsy from last bucardo
Passaged at subconfluency
Fibroblast characterization

10. Procedure Nuclear transfer, fusion, and activation
2 DC pulses of 2.0 kV cm−1for 50 μs each
 M199 with 10% FCS
Ionomycine
Activated oocytes to embryo culture medium I 
Cleaved embryos to embryo culture medium II
Hoechest staining and cell counting
Embryo Transfer
Blastocyst stage
PAG (pregnancy-associated glycoprotein) levels

11. Results One goat carried pregnancy until term PAG levels 70x higher
Microsatelitte analysis
Died minutes after birth to respiratory distress

12. Discussion
Future studies
Ethics
Possible Outcomes

13. Cited Sources
Folch, J. et al. 2009. "First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning." Theriogenology, Volume 71, Issue 6, pp – 1034.
Wani, Nisar Ahmad, et al  "First Cloned Bactrian Camel (Camelus Bactrianus) Calf Produced by Interspecies Somatic Cell Nuclear Transfer: A Step Towards Preserving the Critically Endangered Wild Bactrian Camels." Plos ONE, Volume 12, Issue 5, pp
Manske, Jennifer. “Cloning Endangered Species.” Good For 8, 2001, webphysics.iupui.edu/webscience/bio_archive/goodfor8.html. (Slides 5, 6, 9, 10 Pictures)