Grey wolf genomic history reveals a dual ancestry of dogs.

The grey wolf (Canis lupus) was the first species to give rise to a domestic population, and they remained widespread throughout the last Ice Age when many other large mammal species went extinct. Little is known, however, about the history and possible extinction of past wolf populations or when and where the wolf progenitors of the present-day dog lineage (Canis familiaris) lived1-8. Here we analysed 72 ancient wolf genomes spanning the last 100,000 years from Europe, Siberia and North America. We found that wolf populations were highly connected throughout the Late Pleistocene, with levels of differentiation an order of magnitude lower than they are today. This population connectivity allowed us to detect natural selection across the time series, including rapid fixation of mutations in the gene IFT88 40,000-30,000 years ago. We show that dogs are overall more closely related to ancient wolves from eastern Eurasia than to those from western Eurasia, suggesting a domestication process in the east. However, we also found that dogs in the Near East and Africa derive up to half of their ancestry from a distinct population related to modern southwest Eurasian wolves, reflecting either an independent domestication process or admixture from local wolves. None of the analysed ancient wolf genomes is a direct match for either of these dog ancestries, meaning that the exact progenitor populations remain to be located.

The first evidence for Late Pleistocene dogs in Italy.

The identification of the earliest dogs is challenging because of the absence and/or mosaic pattern of morphological diagnostic features in the initial phases of the domestication process. Furthermore, the natural occurrence of some of these characters in Late Pleistocene wolf populations and the time it took from the onset of traits related to domestication to their prevalence remain indefinite. For these reasons, the spatiotemporal context of the early domestication of dogs is hotly debated. Our combined molecular and morphological analyses of fossil canid remains from the sites of Grotta Paglicci and Grotta Romanelli, in southern Italy, attest of the presence of dogs at least 14,000 calibrated years before present. This unambiguously documents one of the earliest occurrence of domesticates in the Upper Palaeolithic of Europe and in the Mediterranean. The genetic affinity between the Palaeolithic dogs from southern Italy and contemporaneous ones found in Germany also suggest that these animals were an important common adjunct during the Late Glacial, when strong cultural diversification occurred between the Mediterranean world and European areas north of the Alps. Additionally, aDNA analyses indicate that this Upper Palaeolithic dog lineage from Italy may have contributed to the genetic diversity of living dogs.

Effects of low phosphorus supply on the availability of calcium and phosphorus, and musculoskeletal development of growing dogs of two different breeds.

To investigate the impact of a selective reduction in dietary phosphorus (P) supply on healthy growing dogs, a total of 23 Beagles and 30 Foxhound crossbreds (FBI) were used in a feeding trial between 6 and 24 weeks of age. Sixteen Beagles and 19 FBI were fed with selectively reduced P concentrations (low phosphorus, LP). The remaining puppies received a completely balanced control diet (CON). With these diets, the P supply in the Beagles at the age of 12 weeks added up to 2.5 ± 0.6 (LP) and 9.8 ± 1.4 g P/kg DM (CON), and in the FBI 4.3 ± 0.9 (LP) and 13.0 ± 1.6 g P/kg DM (CON). Therefore, the LP Beagles received an average of 33 ± 11% of the recommended daily allowances (RDA) of P, the LP FBI 41 ± 11%. The calcium (Ca) concentration stayed unaltered and led to a Ca/P ratio above the recommended range of 1.3/1 to 2/1. The apparent digestibility (aD) of phosphorus was reduced in the LP Beagle; otherwise, the aD of both minerals was not affected by the P concentration of the diet. The renal excretion of P was reduced to zero in both LP groups while the renal calcium excretion increased significantly. Several of the puppies from both breeds showed impaired appetite, growth, skin and fur quality, and a few also clinically showed relevant signs of a disturbed musculoskeletal system after the LP feeding. A rapid loss of muscle strength and posture within hours led to severe deviation of the limb axis with hyperflexion of the joints but no radiological aberrations or signs of pain. Immediate transition of affected puppies to a balanced diet with sufficient phosphorus resulted in a complete recovery of the puppies in less than one month. The results demonstrate the importance of an adequate P supply on the healthy development of growing dogs.

Evaluation of the in vitro effect of Boldo and Meadowsweet plant extracts on the expression of antimicrobial peptides and inflammatory markers in canine keratinocytes.

Dogs with allergies are prone to skin infections and treatments/preventatives to boost innate immune-defenses are beneficial. The aim of this study was to evaluate the effects of Boldo and Meadowsweet extracts on the expression of ß-defensins (cBD), cathelicidin (cCath), and pro-inflammatory cytokines in canine keratinocyte. This study had two phases. Phase I evaluated mRNA expression of cBD103 and cCath, and secretion of cCath, IL-8 and TNF-α by keratinocytes harvested from healthy (n=5) and atopic (n=5) age-matched beagles exposed to Boldo (2% to 0.2%) and Meadowsweet (1% to 0.2%) extracts. Phase II focused on atopic keratinocytes (n=14) exposed to 0.2% Boldo, 0.2% Meadowsweet, and a mixture of 0.1% of both extracts. Phase I: cBD103 mRNA (all concentrations) and TNF-α secretion (2% Boldo) were increased in atopic compared with healthy keratinocytes. In atopic keratinocytes, cBD103 was increased after exposure to 1.5% and 0.2% Boldo. In healthy keratinocytes, 1% and 0.2% Meadowsweet, and 2% Boldo increased and decreased IL-8 secretion, respectively. In atopic keratinocytes, IL-8 increased after exposure to 1% and 0.4% Meadowsweet extract. Phase II: cBD103 mRNA increased after exposure to 0.2% Meadowsweet and to 0.1% mixture. cCath was increased after 0.2% Boldo, but decreased after 0.2% Meadowsweet or the 0.1% mixture. TNF-α secretion was decreased after 0.2% Boldo. It is concluded that low concentrations of both extracts and their combination may have some effects on cCath and cBD103 without stimulating an inflammatory response. However, more studies are needed to clarify the effects of these extracts on the local immunity.

Nutrigenomic activity of plant derived compounds in health and disease: Results of a dietary intervention study in dog.

The study was conducted to investigate the effects of dietary administrations of four nutraceuticals in dogs. Seventy four dogs were enrolled in the trials, 24 healthy dogs were fed with a control diet (CT) and the experimental groups received for 60days the same diet supplemented with nutraceuticals, namely Echinacea angustifolia (EA, 0.10mg/kg live weight as echinacoside; 14 dogs), Vaccinium myrtillus (VM, 0.20mg/kg live weight as anthocyanidin, 13 dogs), Curcuma longa (CL, 6.60mg/kg live weight as curcumin, 18 dogs with arthrosis), and Sylibum marianum (SM, 1.5mg/kg live weight as sylibin, 8 dogs with hepatopathy). Dogs were weighted at the beginning of study and blood samples were collected at the beginning (T0) and at the end (T60) of the study. VM significantly down regulated TNF, CXCL8, NFKB1 and PTGS2 and decreased plasma ceruloplasmin (CuCp). The activity of EA was evidenced by the significant decrease of TNF and NFKB1 expression and CuCp levels and by the increase of plasma Zn. Administration of CL caused a significant decrease of CuCp and increase of Zn and a down regulation of TNF, CXCL8, NFKB1 and PTGS2, corroborating the anti-inflammatory action of curcuminoids. After 60days of treatment with SM, plasma ALT/GPT activity was reduced and paraoxonase was increased, supporting the antioxidant activity of silymarin, also confirmed by the significant up regulation of SOD2. Results indicated that nutraceutical administrations in dogs can be an interesting approach to modulate immune response in order to improve health condition of animals.

Reference genes for reverse transcription quantitative PCR in canine brain tissue.

BACKGROUND: In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis. RESULTS: Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area. CONCLUSIONS: Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.

Analysis of the canine brain transcriptome with an emphasis on the hypothalamus and cerebral cortex.

The diversity of dog breeds make the domestic dog a valuable model for identifying genes responsible for many phenotypic and behavioral traits. The brain, in particular, is a region of interest for the analysis of molecular changes that are involved in dog-specific behavioral phenotypes. However, such studies are handicapped due to incomplete annotation of the dog genome. We present a high-coverage transcriptome of the dog brain using RNA-Seq. Two areas of the brain, hypothalamus and cerebral cortex, were selected for their roles in cognition, emotion, and neuroendocrine functions. We detected many novel features of the dog transcriptome, including 13,799 novel exons, 51,357 exons with unique 5' or 3' modifications, and many novel alternative splicing events. We provide some examples of novel features in genes that are related to domestication, including ADCY8, SMOC2, and PRNP. We also found 247 novel protein-coding genes and 328 noncoding RNAs, including 57 long noncoding RNAs that represent the first empirical evidence for a large fraction of noncoding RNAs in the dog. In addition, we analyze both gene expression and alternative splicing differences between the hypothalamus and cerebral cortex and find that there is very little overlap between genes that are differentially alternatively spliced and genes that are differentially expressed. We thereby suggest that researchers who want to pinpoint the genetic causes for dog breed-specific traits and diseases should not confine their studies to gene expression alone, but should consider other factors such as alternative splicing and changes in untranslated regions.

Heterologous expression and functional characterization of matrix metalloproteinase-11 from canine mammary tumor.

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.

A thyroid peroxidase (TPO) mutation in dogs reveals a canid-specific gene structure.

Congenital hypothyroidism with goiter (CHG) occurring as an autosomal recessive disorder is typically due to a defect of thyroid hormone synthesis (aka dyshormonogenesis). Thyroid peroxidase (TPO) is a multifunctional, heme-containing enzyme whose activity is required, and several inactivating TPO mutations causing CHG in humans and dogs have been described. Recently, two half-sib Spanish water dog (SWD) pups were diagnosed with CHG based on clinical signs, endocrine testing, and thyroid histology. TPO enzyme activity was absent, and immuno-cross-reactive TPO was undetectable in affected-dog thyroid tissue. A single guanosine insertion was observed in the first exon of the affected-dog TPO cDNA at a site not previously thought to be within the coding sequence. The insertion allele segregated with the deduced disease allele in the SWD breed and was not observed in unrelated dogs of various breeds. Comparison of the insertion site (an 8-nt poly-G tract) with the orthologous sequences of other mammalian reference genomes revealed that the octa-G tract obliterated the intron 1 splice acceptor site and the exon 2 translation initiation codon found at that position in other species. An in-frame ATG in strong Kozak consensus context was observed in the normal dog sequence 12 codons 5' of the usual mammalian start site, suggesting that dogs have lost the noncoding exon 1 demonstrated in human and mouse. A survey of TPO sequences in other carnivore species indicates that the poly-G tract necessitating an alternative translation initiation site is a canid-specific feature.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Evaluation of breed effects on n-3 PUFA metabolism with dietary flaxseed oil supplementation in dogs.

PUFA are important for human and animal health. To our knowledge, previous studies investigating the metabolism of PUFA in dogs have not examined breed differences. The aim of the present study was to evaluate the potential to elongate PUFA in two pure breeds of dogs. Plasma fatty acid composition (%) was measured in dogs during 3 weeks supplementation with flaxseed oil (57 % α-linolenic acid (ALA) and 17 % linolenic acid (LA)) at the rate of 100 ml/kg food following 4 months of feeding an identical standard basal diet. Plasma extracted at fasting state from five beagles and five greyhounds was analysed by GC. Plasma ALA, EPA and LA increased steadily and significantly from days 0 to 22 (P < 0.05); however, no significant breed differences were shown. Plasma DHA levels, on the other hand, showed no significant increase over time, but a significant breed difference was observed, with beagles having higher plasma level from day 0 (P = 0.002). This breed difference requires further investigation. Levels of ALA and EPA were still rising significantly between days 15 and 22, indicating that PUFA levels in plasma had not stabilised in 3 weeks. These findings together suggest that flaxseed oil could be a useful source of PUFA in dogs, especially ALA and EPA, and that breed differences may be important.

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins.

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.

Characterization of canine BCL6 cDNA and detection systems for its protein expression.

BCL6 is known to be a key molecule in germinal center (GC) formation of lymph nodes, and its expression profiles have been implicated in the prognosis of diffuse large B-cell lymphoma in humans. The present study was carried out to characterize canine BCL6 cDNA and to indicate the technical methods for detection of the BCL6 protein in dog tissues. The deduced amino acid sequence of canine BCL6 showed close homology to that of human BCL6 (96.3%), especially in the zinc-finger motifs and POZ (poxvirus and zinc finger) domain with complete identity. Immunoblot analysis of a canine lymph node with an anti-human BCL6 monoclonal antibody revealed a band of 80 kDa. Immunohistochemical staining using the same antibody produced positive reactions in the cells exclusively localized in the GC of a canine lymph node. This study will be useful for the molecular classification of canine B-cell lymphomas with different prognoses.

Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.

Caspases-1, 4, 5, and 12 and other proteins containing caspase recruitment domains (CARDs) play crucial roles in the induction of inflammatory processes. Recently, hybrid caspase-1/4 mRNAs encoding proteases with two CARDs were identified in cat and dog, indicating that the molecular machinery of caspase-dependent inflammation has an unconventional composition in members of the order Carnivora. Here we extended these studies and identified, both in cat and dog, splice variants of caspase-12, which also contained two CARDs. Comparative genomics analysis of the repertoire of canine CARD proteins revealed that the gene encoding NLRC4/IPAF, which is implicated in the inflammatory response to cytosolic flagellin, was inactivated by deleterious mutations in the dog. Our results demonstrate that the repertoires of CARD proteins in cat and dog differ significantly from that of humans and suggest the existence of uncharacterized pathways of inflammasome-mediated signaling in Carnivora.

Pharmacogenetic and metabolic differences between dog breeds: their impact on canine medicine and the use of the dog as a preclinical animal model.

There is limited information describing species related pharmacogenetic differences in animals. Despite the lack of genetic information in veterinary medicine, breed specific responses to endogenous and exogenous substances have been reported across many species. This finding underscores the importance of obtaining insight into the genotypic and phenotypic variation present across breeds. This article provides a summary of the literature pertaining to canine breed differences in physiology, drug response, drug pharmacokinetics, and metabolic idiosyncrasies. The existing knowledge of pedigrees and the known phenotypes and genotypes of dogs provides important information for determining mode of inheritance, penetration, and other major characteristics of heritable traits. Understanding these breed differences will improve canine population predictions (for canine drug products) and may be of value when extrapolating toxicology data from dogs to humans.

Canine tyrosine hydroxylase (TH) gene and dopamine beta -hydroxylase (DBH) gene: their sequences, genetic polymorphisms, and diversities among five different dog breeds.

Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.

Mitochondrial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves.

The question of the origins of the dog has been much debated. The dog is descended from the wolf that at the end of the last glaciation (the archaeologically hypothesized period of dog domestication) was one of the most widespread among Holarctic mammals. Scenarios provided by genetic studies range from multiple dog-founding events to a single origin in East Asia. The earliest fossil dogs, dated approximately 17-12,000 radiocarbon ((14)C) years ago (YA), were found in Europe and in the Middle East. Ancient DNA (a-DNA) evidence could contribute to the identification of dog-founder wolf populations. To gain insight into the relationships between ancient European wolves and dogs we analyzed a 262-bp mitochondrial DNA control region fragment retrieved from five prehistoric Italian canids ranging in age from approximately 15,000 to approximately 3,000 (14)C YA. These canids were compared to a worldwide sample of 547 purebred dogs and 341 wolves. The ancient sequences were highly diverse and joined the three major clades of extant dog sequences. Phylogenetic investigations highlighted relationships between the ancient sequences and geographically widespread extant dog matrilines and between the ancient sequences and extant wolf matrilines of mainly East European origin. The results provide a-DNA support for the involvement of European wolves in the origins of the three major dog clades. Genetic data also suggest multiple independent domestication events. East European wolves may still reflect the genetic variation of ancient dog-founder populations.

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1).

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.