Cloning of an Endangered Species (Bos gaurus) Using Interspecies Nuclear Transfer

Abstract

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan Ͻ 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations. 79

Mammalian nuclear transfer

“…Using antibodies against 5-methyl cytosine, two independent studies showed that the cloned bovine embryos did not undergo normal global demethylation in early embryogenesis and even showed precocious de novo methylation , with a Since the generation of "Dolly," a wide range of mammals have been used for nuclear transfer (NT) and produced live born clones. Interspecies NT (Bos gaurus donor nuclei into domestic cow oocytes) was used to demonstrate the potential to rescue endangered species (only late-stage pregnancies were demonstrated in this report [Lanza et al, 2000b]). a Cultured donor cells were in most cases synchronized (G0/G1) either by serum starvation or contact inhibition before nuclear transfer (NT).…”

“…This approach was successfully used to produce transgenic sheep, pigs, and cows (reviewed in Campbell et al, 2005). Furthermore, the possibility to rescue endangered species (Bos gaurus) by interspecies NT using domestic animals as oocyte donors has been reported indicating another potentially useful application of NT (Lanza et al, 2000b). It should be emphasized that the abnormalities of cloned animals are likely not problematic for propagating livestock animals, because epigenetic abnormalities are lost in the offspring of the clones (Tamashiro et al, 2002;Jaenisch, 2004).…”

Des ovules en abondance ?

“…Une autre perspective s'apparente plutôt au clonage interspé-cifique et consisterait à introduire un noyau somatique humain dans un ovocyte de femelle animale, en espérant réaliser un ovule chimérique à l'issue de la méiose in vitro. Ce bricolage a été réalisé avec succès, dans le cadre de la préservation d'animaux menacés, pour des espèces très proches: croisements mouflon/mouton [23] ou bovin sauvage (Bos javanicus)/bovin domestique (Bos taurus) [24]. Des auteurs chinois ont même tenté de conserver ainsi le panda géant, mais leur expérience audacieuse (génome de panda introduit dans l'ovule de lapin et gestation de l'embryon chimérique assurée par une chatte…) n'a pas conduit à la naissance, bien que le dévelop-pement post-implantatoire, chez la chatte, de clones interspé-cifiques panda-lapin soit rapporté [25].…”

A protocol for the cryoconservation of breeds by low-cost emergency cell banks – a pilot study

“…A somatic cell bank consists of ear-tag tissues, which can be preserved in liquid nitrogen (LN) and subsequently used as donor cells for animal cloning. During the last decade, the technique of reproductive somatic nuclear transfer (NT) was successfully applied in livestock (sheep, cattle, goats, pigs, horses), rodents (mice, rats) and pet animals (cats, dogs), as reviewed in Kues and Niemann (2004), and also in the preservation of endangered breeds (Lanza et al, 2000;Wells et al, 1998;White et al, 1999) and wild animal species (Loi et al, 2001). The best success rates in terms of live offspring so far were obtained with cloned cattle and goats.…”