Arising from: B. C. Lee et al. Nature 436, 641 (2005); see also communication from the H. G. Parker, L. Kruglyak and E. A. Ostrander
Somatic-cell nuclear-transfer technology has been used to clone a variety of animal species1,2,3, but the overall efficiency of the cloning process and the viability of embryos has remained low4. Until Lee et al. described the cloning of two Afghan hounds by nuclear transfer from adult skin fibroblasts into oocytes that had matured in vivo5, dog cloning had been unsuccessful because of the difficulty of collecting canine oocytes matured in vivo at metaphase II (ref. 6). Here we provide independent evidence from the Seoul National University Investigation Committee that Snuppy, the one of the pair to survive, is a genuine clone.
To investigate whether the cloned dog was genetically identical to the donor Afghan, we obtained blood samples from Snuppy, from the male Afghan hound that provided the somatic cell, and from the surrogate mother. In addition, autopsy samples from the since-deceased mixed-breed dog that originally provided the egg used to create Snuppy were obtained from the research team who generated the cloned dog. DNA was extracted from the blood and autopsy samples and used for microsatellite analysis of genomic DNA and nucleotide sequences of mitochondrial DNA.
The microsatellite analyses were performed with genomic DNA from four dogs (Snuppy, the donor Afghan, the surrogate mother and the egg donor) using 27 canine-specific markers that allow extremely inbred animals to be distinguished. As shown in Table 1, which shows only 8 of 27 loci used for microsatellite analysis, Snuppy and the donor Afghan have identical microsatellite patterns for all loci, whereas the surrogate mother evidently has a different genetic origin.
For 18 of the microsatellite loci analysed (including those shown in Table 1), the egg donor and Snuppy showed distinct microsatellite patterns. The microsatellite loci used for the analyses show enough variation among Afghan hound individuals to allow them to be distinguished (results not shown). Thus, the Committee's microsatellite analysis of genomic DNA demonstrates that the cloned dog Snuppy is genetically identical to its fibroblast-donor Afghan hound.
If Snuppy is a cloned dog, his mitochondrial DNA (mtDNA) should be identical to that of the egg donor, but not to that of the fibroblast-donor Afghan. To test this, we determined partial sequences of four regions of the dog mtDNA. Based on the complete nucleotide sequence (GenBank accession number U96639), four primer sets were synthesized and used for polymerase chain reaction (PCR)7: the cytochrome b gene (L14,252–L14,631), the 16S ribosomal RNA gene (L2,033–L2,472), and the two overlapping hypervariable D-loop regions (L15,622–L16,030 and L15,485–L16,090). PCR products were sequenced and a BLAST search confirmed their identity as mtDNA.
The sequence alignments of 409 base pairs of the hypervariable D-loop region revealed eight mismatches between the mtDNA of Snuppy and the donor Afghan (Table 2). A non-match was observed even in more conserved regions (one mismatch in the cytochrome b gene and two mismatches in the 16S rRNA gene). These results indicate that Snuppy could not have been produced by splitting the early blastomere from which the donor Afghan originated. The sequence alignments also revealed a perfect match between Snuppy and the egg donor, and a non-match between Snuppy and the surrogate mother (Table 2). This is consistent with Snuppy being a nuclear clone of the donor Afghan.
On the basis of the results of both a microsatellite analysis of genomic DNA and a sequence comparison of mtDNA, it is highly unlikely that Snuppy came either from extreme inbreeding or from blastomere separation (also known as twinning). It is virtually certain that Snuppy was generated from somatic-cell nuclear transfer, as claimed by Lee et al.5.