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Based on the physiologically non-self-renewing property of articular cartilage, the treatment of articular cartilage lesions remains an important challenge. Articular cartilage, the target of osteoarthritis (OA), contains renewable cartilage stem/progenitor cells (CSPCs) to maintain the tissue homeostasis. Compared with other adult stem cells, the self-renewal capacity and preference for chondrogenesis of resident CSPCs have propelled their exciting therapeutic prospects for cartilage repair. Tibetan mastiff, the ancestor of large breed dogs worldwide, represents a valuable model for human diseases. To our knowledge, this is the first study to capture the emergence of Tibetan mastiff-derived CSPCs. Our study aimed to unveil the biological properties and differentiation multipotency of CSPCs. CSPCs were characterized by the clonogenicity, growth curves and karyotype analysis, respectively. The results showed that the CSPCs of Tibetan mastiff displayed self-renewal ability, proliferative potential, and hereditary stability. Moreover, the CSPCs of Tibetan mastiff also positively expressed recognized surface antigens of human CSPCs. Additionally, CSPCs exhibited differentiation multipotency, including osteogenic, adipogenic and chondrogenic potential in vitro. These findings may propell CSPCs application in a clinical study, suggesting potential therapeutic treatment for OA or related joint diseases. [ABSTRACT FROM AUTHOR]

In November 1828, King George IV presented to the menagerie of the Zoological Society of London a pair of Tibetan Mastiffs (Canis lupus familiaris) sent from Nepal by Brian Houghton Hodgson, the Assistant Resident at the British Residency in Kathmandu. The dogs' stay was brief: within weeks of their arrival, both dogs had succumbed to distemper, a disorder which periodically ran rampant through the Society's collections. However, during their short lives they caught the imagination of those naturalists fortunate enough to see them, and they live on in a number of watercolours commissioned by Hodgson before they started on their journey to Britain. This paper traces the brief existence of the "Thibet watch dogs" in London, paying close attention to their intended and abortive use as a gift to gain favour with the King, and places their portraits within the wider context of Hodgson's work in Nepal. [ABSTRACT FROM AUTHOR]

A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb–O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the β-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked β-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the β-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb–O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed's relatively short duration of residency at high altitude. [ABSTRACT FROM AUTHOR]

The control region of mitochondrial DNA (mtDNA) was obtained from 40 purebred Chinese Tibetan Mastiffs (TMs). Sequence structure and genetic diversity were analyzed, and a phylogenetic tree was constructed. The TM mtDNA control region was composed of ETAS (extended termination associated sequences), CD (a central domain) and CSBs (conserved sequenced blocks) and sequence length showed some diversity, which was mainly caused by the number of 10 nucleotide repeat units [5′-GTA CAC GT (G/A) C-3′] between CSB I and CSB II, which ranged from 27 to 35 among individuals. Seventy-five polymorphic sites were identified, which defined 37 haplotypes; the haplotype diversity was 0.990, and the nucleotide diversity was 1.201. Based on the control region sequences, Chinese TMs were divided into three categories, which were consistent with the origin and geographical classification of TMs. Phylogenetic analysis of 538-bp HVR-I sequences revealed that TMs were most closely related to Labrador Retrievers. [ABSTRACT FROM AUTHOR]

Bone marrow mesenchymal stem cells (BM‑MSCs) are pluripotent stem cells that are regarded as ideal resources for the reconstruction of tissues and organs. The Tibetan mastiff is a breed of domesticated Chinese native dog that is well‑adjusted to the high‑altitude environments of Tibet. To the best of our knowledge, the biological characterization and multi‑lineage differentiation of Tibetan mastiff BM‑MSCs have not been reported previously. Therefore, the present study aimed to investigate the biological characteristics and therapeutic potential of Tibetan mastiff BM‑MSCs. A cell culture system was constructed and cells were cultured to 23 passages in vitro. Growth curves and colony formation studies suggested that BM‑MSCs had a high self‑renewal capacity and that their proliferation rate declined with age. Karyotype analysis demonstrated that BM‑MSCs were diploid and genetically stable. Semi‑quantitative polymerase chain reaction analysis revealed that BM‑MSCs positively expressed cluster of differentiation (CD)73, CD90, CD105, CD166 and vimentin, although they were negative for the endothelial cell marker CD31. Additionally, immunofluorescence staining revealed that the cells expressed CD29, CD44, CD90, CD105 and vimentin. Flow cytometric analysis revealed that the rates of positive expression of vimentin, CD44, CD90 and CD105 were all >97%. BM‑MSCs were able to differentiate into adipocytes, osteoblasts, cartilage cells, hepatocytes and functional insulin‑secreting cells. In conclusion, Tibetan mastiff BM‑MSCs may be purified successfully using a whole bone marrow culture method. The findings of the current study suggested important potential applications of BM‑MSCs as a source for regenerative therapies. [ABSTRACT FROM AUTHOR]

Canine parvovirus 2 (CPV-2) was first identified in 1978, and is responsible for classic parvoviral enteritis. Despite the widespread vaccination of domestic carnivores, CPVs have remained important pathogens of domestic and wild carnivores. In this study, we isolated CPV-2 from Tibetan mastiffs and performed a global analysis of the complete VP2 gene sequences of CPV-2 strains in China. Six isolates were typed as new CPV-2a, according to key amino acid positions. On a phylogenetic tree, these six sequences formed a distinct clade. Five isolates occurred on the same branch as KF785794 from China and GQ379049 from Thailand; CPV-LS-ZA1 formed a separate subgroup with FJ435347 from China. One hundred ninety-eight sequences from various parts of China and the six sequences isolated here formed seven distinct clusters, indicating the high diversity of CPVs in China. Of 204 VP2 sequences, 183 (91.04%) encoded the mutation Ser297Ala, regardless of the antigenic type, implying that most Chinese CPV-2 strains contain the VP2 mutation Ser297Ala. However, the biological significance of this change from prototype CPV-2a/2b to new CPV-2a/2b types remains unclear. This study is the first to isolate new CPV-2a from the Tibetan mastiff. Our data show that new CPV-2a/2b variants are now circulating in China.

The Tibetan Mastiff (TM), a native of the Tibetan Plateau, has quickly adapted to the extreme highland environment. Recently, the impact of positive selection on the TM genome was studied and potential hypoxia-adaptive genes were identified. However, the origin of the adaptive variants remains unknown. In this study, we investigated the signature of genetic introgression in the adaptation of TMs with dog and wolf genomic data from different altitudes in close geographic proximity. On a genome-wide scale, the TM was much more closely related to other dogs than wolves. However, using the 'ABBA/BABA' test, we identified genomic regions from the TM that possibly introgressed from Tibetan gray wolf. Several of the regions, including the EPAS1 and HBB loci, also showed the dominant signature of selective sweeps in the TM genome. We validated the introgression of the two loci by excluding the possibility of convergent evolution and ancestral polymorphisms and examined the haplotypes of all available canid genomes. The estimated time of introgression based on a non-coding region of the EPAS1 locus mostly overlapped with the Paleolithic era. Our results demonstrated that the introgression of hypoxia adaptive genes in wolves from the highland played an important role for dogs living in hypoxic environments, which indicated that domestic animals could acquire local adaptation quickly by secondary contact with their wild relatives. [ABSTRACT FROM AUTHOR]

In this study, the complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined, and the phylogenetic relationships between the Tibetan Mastiff and other species of Canidae were analyzed using the coyote (Canis latrans) as an outgroup. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16 710 bp, and included 22 tRNA genes, 2S rRNA gene, 13 protein-coding genes and one non-coding region (D-loop region), which is similar to other mammalian mitochondrial genomes. The characteristics of the protein-coding genes, non-coding region, tRNA and rRNA genes among Canidae were analyzed in detail. Neighbor-joining and maximum-parsimony trees of Canids constructed using 12 mitochondrial protein-coding genes showed that as the coyotes and Tibetan wolves clustered together, so too did the gray wolves and domestic dogs, suggesting that the Tibetan Mastiff originated from the gray wolf as did other domestic dogs. Domestic dogs clustered into four clades, implying at least four maternal origins (A to D). The Tibetan Mastiff, which belongs to clade A, appears to be closely related to the Saint Bernard and the Old English Sheepdog.

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.

Tibetan Mastiff is one of the most archaic, ferocious and the largest dogs in the world. A total of 140 individuals from four geographically separated populations in China (Tibet, Gansu, Qinghai and Beijing) were sampled and genetic diversity was assessed using 10 microsatellite loci on eight different chromosomes. The mean number of alleles per locus ranged from 6 to 13. The mean observed and expected heterozygosities, polymorphism information content and allelic richness were 0.69, 0.79, 0.76 and 7.59, respectively, indicating relatively high genetic diversity in Tibetan Mastiff. However, a highly significant deficiency in heterozygote was observed within populations (mean FIS = 0.11, bootstrap 95% confidence interval (0.06, 0.17)) and total inbreeding (mean FIT = 0.12, bootstrap 95% confidence interval (0.06, 0.18)), along with strong inbreeding coefficients within populations (Fis > 0.09), all of which suggested that intense inbreeding practices occurred in Tibetan Mastiff. Therefore, effective and appropriate breeding management projects in present Tibetan Mastiff will be desirable and urgent. Low genetic differentiation was obtained with a mean FST of 0.01 (bootstrap 95% confidence interval (0.007, 0.019)). Additionally, the four Tibetan Mastiff populations showed close relationships in the neighbor-joining polygenetic tree based on the coancestral genetic distances. Tibetan Mastiff was investigated by using microsatellite loci at the first time, which could facilitate the better understanding of present situation at the molecular level, breed conservation and utilization in Tibetan Mastiff.

Tibetan Mastiff has adapted to the extreme environment of the Qinghai-Tibetan Plateau. Yet, the underlying mechanisms of its high-altitude-adaptation and origin remains elusive. Here, we generated the draft genomes of Mongolia Mastiff, Tibetan Mastiff, and Canis Lupus. The phylogenetic tree uncovered that Tibetan Mastiff and Mongolia Mastiff were derived from Canis Lupus species. The comparative genomic analyses identified that the expansion of gene families related to DNA repair and damage response, and contraction related to ATPase activity revealed the genetic adaptations of Tibetan Mastiff and Canis Lupus to high altitude. In addition, the Tibetan Mastiff and Canis Lupus had signals of positive selection for genes involved in fatty-acid α/β- oxidation for highland adaptation. Notably, the positively selected TERT of Tibetan Mastiff should be an adaptive trait for correcting DNA damage. These findings suggested that the Tibetan Mastiff and Canis Lupus evolves basic strategies for adaptation to high altitude. • De novo assembled the genome sequence of Mongolia mastiff, Tibetan mastiff, and Canis Lupus with excellent continuity at the contig and scaffold levels. • Tibetan Mastiff and Mongolia Mastiff were derived from Canis Lupus. • The expansion of gene families related to DNA repair and damage response, as well as the contraction related to ATPase activity revealed the genetic adaptations of Tibetan Mastiff and Canis Lupus to high altitude. • The Tibetan Mastiff and Canis Lupus had signals of positive selection for genes involved in fatty-acid α/β-oxidation for highland adaptation. Notably, the positively selected TERT of Tibetan Mastiff should be an adaptive trait for correcting DNA damage. [ABSTRACT FROM AUTHOR]

With the assistance of their human companions, dogs have dispersed into new environments during the expansion of human civilization. Tibetan Mastiff (TM), a native of the Tibetan Plateau, was derived from the domesticated Chinese native dog and, like Tibetans, has adapted to the extreme environment of high altitude. Here, we genotyped genome-wide single-nucleotide polymorphisms (SNPs) from 32 TMs and compared them with SNPs from 20 Chinese native dogs and 14 gray wolves (Canis lupus). We identified 16 genes with signals of positive selection in the TM, with 12 of these candidate genes associated with functions that have roles in adaptation to high-altitude adaptation, such as EPAS1, SIRT7, PLXNA4, and MAFG that have roles in responses to hypoxia. This study provides important information on the genetic diversity of the TM and potential mechanisms for adaptation to hypoxia. [ABSTRACT FROM PUBLISHER]

Abstract: Ten 3-month-old Tibetan mastiffs became ill 2 days after they were bought from a Tibetan mastiff exhibition, and 4 of them died 2 weeks later. A canine influenza virus (ZJ0110) was isolated from the lung of a deceased Tibetan mastiff and was characterized in detail. Sequence analysis indicated that the 8 genes of the canine isolate were most similar to those of avian-origin canine influenza viruses (H3N2) isolated in South Korea in 2007, with which they shared >98% sequence identity. ZJ0110 could experimentally infect 6-month-old beagles by intranasal inoculation and by airborne transmission, causing severe respiratory syndrome. Moreover, ZJ0110 could replicate in the upper respiratory tracts of mice and guinea pigs, and the virus titer was comparable to that in the upper respiratory tracts of dogs. Although the virus was genetically of avian origin, ZJ0110 could not experimentally infect chicken or ducks by intranasal inoculation. These results suggest that dogs might be an intermediary host in which avian influenza viruses adapt to replicate in mammals. [Copyright &y& Elsevier]

Many studies have been reported the origin and evolution of domesticated animals, particularly dogs, however, few have systematically examined the Tibetan Mastiff (TM), a dog that lives primarily in the Qing-Tibet Plateau. Here, we study the origin and evolution of TM based on the population comparison of mtDNA D-loop sequences from TM and other dogs across the world. Intriguingly, non-simultaneous arrivals of Tibetan Mastiffs ancestors to Tibet occurred, which may result from continuous Tibetans' settlement on Qing-Tibet Plateau supported by archaeological and genetic evidence. High genetic and haplotype diversity and robust haplotypes sharing with other dogs unique to East Asia are observed in Tibetan Mastiff, supporting that it is a very archaic breed derived probably from East Asia. [ABSTRACT FROM AUTHOR]

Abstract: Background: Toxoplasma gondii is an amphixenosis which has extensive hosts. In recent years, the prevalence of T. gondii in China has been reported, while little is known on the survey of T. gondii infection in northwest China, especially in yaks (Bos grunniens) and Tibetan mastiffs (Canis lupus familiaris). The current study survey the infection of T. gondii in Tibetan mastiffs and yaks in Qinghai Province, China. Methods: The indirect hemagglutination test (IHAT) was used to examine T. gondii antibodies in 1 795 serums, including 192 Tibetan mastiffs and 1603 yaks in Qinghai Province, China. Results: In this study, the seroprevalence of T. gondii infection was 8.52%. Twenty (10.42%) of 192 serums of Tibetan mastiffs and 133 (8.30%) of 1603 serums of yaks were seropositive. The seroprevalence of T.gondii infection in Tibetan mastiffs in breeding farm (1.08%) was lower than that in the field (19.19%), and the difference was statistically significant (P <0.05). The seroprevalence of antibodies to T.gondii in yaks ranged from 5.45% to 13.28% among the four different areas. The seroprevalence in different age groups were determined with apparent association. Conclusions: The results indicated that T.gondii infection was prevalent in Tibetan mastiffs and yaks, which have implications for public health in this region. To our knowledge, this is the first seroprevalence survey of Tibetan mastiffs infected by T. gondii in The People's Republic of China [ABSTRACT FROM AUTHOR]

This test analyzes the Tibetan mastiff cashmere physical and chemical properties of cashmere fiber, such as fiber length, fineness, crimp degree and strength. Through the technology of cotton spinning equipment, semi-worsted cashmere yarn of Tibetan mastiff was spun. Tibetan mastiff cashmere and wool have many similar characteristics, can be used as new textile raw materials to develop new products. Because of with natural color and luster, its products can stain so without post-processing techniques, which has reduced energy consumption to reduce the role of chemical pollution, has construction of the green industry on the practical significance. The development of Tibetan mastiff cashmere fibers created new values for the mastiff farming. [ABSTRACT FROM AUTHOR]

Tibetan Mastiff is one of the most archaic, ferocious and the largest dogs in the world. A total of 140 individuals from four geographically separated populations in China (Tibet, Gansu, Qinghai and Beijing) were sampled and genetic diversity was assessed using 10 microsatellite loci on eight different chromosomes. The mean number of alleles per locus ranged from 6 to 13. The mean observed and expected heterozygosities, polymorphism information content and allelic richness were 0.69, 0.79, 0.76 and 7.59, respectively, indicating relatively high genetic diversity in Tibetan Mastiff. However, a highly significant deficiency in heterozygote was observed within populations (mean FIS= 0.11, bootstrap 95% confidence interval (0.06, 0.17)) and total inbreeding (mean FIT = 0.12, bootstrap 95% confidence interval (0.06, 0.18)), along with strong inbreeding coefficients within populations (Fis>0.09), all of which suggested that intense inbreeding practices occurred in Tibetan Mastiff. Therefore, effective and appropriate breeding management projects in present Tibetan Mastiff will be desirable and urgent. Low genetic differentiation was obtained with a mean FST of 0.01 (bootstrap 95% confidence interval (0.007, 0.019)). Additionally, the four Tibetan Mastiff populations showed close relationships in the neighbor-joining polygenetic tree based on the coancestral genetic distances. Tibetan Mastiff was investigated by using microsatellite loci at the first time, which could facilitate the better understanding of present situation at the molecular level, breed conservation and utilization in Tibetan Mastiff. [ABSTRACT FROM AUTHOR]

The Hequ Tibetan MastiffCanis lupus familiaris(Carnivora: Canidae) is a primitive breed of large dogs native to the northeastern Qinghai-Tibetan Plateau of China. In this study, its complete mitochondrial genome sequence has been assembled and characterized using high-throughput Illumina sequencing technology. The circular genome is 16 730 bp in length, and possesses all genomic components as typically found in most other metazoan mitogenomes. The gene arrangement is identical to those of most other vertebrates. Except forND4Lwith GTG as its start codon, all the other PCGs are initiated with an ATR (ATA/ATG) codon. Three distinct stop codons are employed, i.e. AGA forCYTB, TAA forATP6, ATP8, COX1, COX2, ND1, ND4L, ND5andND6, and an incomplete stop codon T forCOX3, ND2, ND3andND4. The nucleotide composition is asymmetric (31.6% A, 25.5% C, 14.2% G and 28.7% T) with an overall A + T content of 60.3%. These data would contribute to our better understanding its evolutionary history. [ABSTRACT FROM AUTHOR]

In this study, the complete mitochondrial genome sequence of the Tibetan Mastiff was reported. The total length of the mitogenome is 16,729 bp. It contains the typical structure, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region is in line with other canine animals. We further identified genome-wide variations among different canine mitochondrial genomes and indicated that the D-loop region harbors the most sequence variation, which will provide sequence variation information for the protection and utilization of the Tibetan Mastiff germplasm resource. [ABSTRACT FROM AUTHOR]

The Tibetan Mastiff (TM), a native of the Tibetan Plateau, has quickly adapted to the extreme highland environment. Recently, the impact of positive selection on the TM genome was studied and potential hypoxia-adaptive genes were identified. However, the origin of the adaptive variants remains unknown. In this study, we investigated the signature of genetic introgression in the adaptation of TMs with dog and wolf genomic data from different altitudes in close geographic proximity. On a genome-wide scale, the TM was much more closely related to other dogs than wolves. However, using the 'ABBA/BABA' test, we identified genomic regions from the TM that possibly introgressed from Tibetan gray wolf. Several of the regions, including the EPAS1 and HBB loci, also showed the dominant signature of selective sweeps in the TM genome. We validated the introgression of the two loci by excluding the possibility of convergent evolution and ancestral polymorphisms and examined the haplotypes of all available canid genomes. The estimated time of introgression based on a non-coding region of the EPAS1 locus mostly overlapped with the Paleolithic era. Our results demonstrated that the introgression of hypoxia adaptive genes in wolves from the highland played an important role for dogs living in hypoxic environments, which indicated that domestic animals could acquire local adaptation quickly by secondary contact with their wild relatives., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al.(2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al.(2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author. [ABSTRACT FROM AUTHOR]