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

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

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)

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

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

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

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

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

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

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

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

Discussion Future studies Ethics Possible Outcomes

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. 1026 – 1034. Wani, Nisar Ahmad, et al. 2017. "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. 1-11. Manske, Jennifer. “Cloning Endangered Species.” Good For 8, 2001, webphysics.iupui.edu/webscience/bio_archive/goodfor8.html. (Slides 5, 6, 9, 10 Pictures)