Endangered Wolves Cloned from Adult Somatic Cells

Abstract

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

Conservation of the Sapsaree (Canis familiaris), a Korean Natural Monument, using Somatic Cell Nuclear Transfer

“…Furthermore, their mitochondrial (mt) DNA originated from the oocytes of donor dogs. This result is in line with our previous studies [9,14]. In regard to mtDNA, it is important to note that it is believed that the mtDNA of offspring is transmitted from the mother, not the father.…”

“…Additionally, female gray wolves (Canis lupus) were cloned from a 2-year-old female wolf [9], and male cloned gray wolves from postmortem tissue [14]. This indicates that canine SCNT can be applied to conserve endangered species of the Canidae family.…”

“…Some of the trials to conserve endangered animals using intra-and interspecies SCNT have failed to produce viable offspring [2,11,18]. However, live cloned offspring, such as normal mouflon [13], african wild cat [4,5] and gray wolves [9,14], have been obtained successfully using SCNT.…”

“…This study was conducted in accordance with recommendations described in "The Guide for the Care and Use of Laboratory Animals" published by Seoul National University. The surgical approach for collection of in vivo matured oocytes and transfer of cloned embryos to the oviduct was performed in accordance with our studies [7,9,12,14]. In brief, anesthesia was induced with 6 mg/kg ketamine HCl and 1 mg/kg xylazine, and general anesthesia was maintained with 2% isoflurane.…”

Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs

“…However, this methodology results in the production of nuclear-cytoplasmic hybrids [43,44]. Despite numerous attempts in a wide variety of species, the number of live births of cloned offspring is still limited to instances that combine genetic compartments of closely related species, as observed in gaur-bovine [45], mouflonsheep [46], African wild cat-domestic cat [47] and gray wolf-domestic dog experiments [48]. In most cases, interspecies/intergeneric SCNT (iSCNT) embryos developed to the blastocyst stage.…”