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You are here: Home / Publications / Bibliographies and Resource Guides / Canine Models in Biomedical Research, 1990-2009  / Molecular Genetics  Printer Friendly Page
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Canine Models in Biomedical Research,  1990-2009
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Molecular Genetics

Baker, S.M., L. Karlsson, and R.L. Thurmond (2003). Cloning, expression, purification, and activity of dog (canis familiaris) and monkey (saimiri boliviensis) cathepsin s. Protein Expression and Purification 28(1): 93-101. ISSN: 1046-5928.
NAL Call Number: QP551.P695818
Abstract: Cathepsin S is the key protease responsible for the removal of the invariant chain from MHC class II molecules and, as such, has attracted much attention as a target for developing immunosuppressive drugs. To help in testing candidate compounds, the monkey (Saimiri boliviensis) and dog (Canis familiaris) cathepsin S cDNAs have been cloned. The monkey cDNA sequence encodes a 330 amino acid protein with 95% homology to human cathepsin S. The dog cDNA sequence encodes a 331 amino acid protein with 91% homology to human cathepsin S. The amino acid differences do not have a major effect on the hydrolysis of the substrate Z-VVR-AMC, but may affect the substrate specificity. As for human and bovine cathepsin S, activity against Z-VVR-AMC extends into the neutral pH range. These parameters are important for understanding the role of cathepsin S in different species and for testing inhibitors in animal models of autoimmunity.
Descriptors: enzymology, biochemistry and molecular biophysics, genetics, immune system, chemical coordination and homeostasis, methods and techniques, amino acid sequence, peptide sequence, autoimmunity .

Beard, B.C., K.A. Keyser, G.D. Trobridge, L.J. Peterson, D.G. Miller, M. Jacobs, R. Kaul, and H.P. Kiem (2007). Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, or foamy virus. Human Gene Therapy 18(5): 423-34. ISSN: 1043-0342.
Abstract: Recent advances have allowed for improved retrovirus-mediated gene transfer, and therapeutic benefits have been described in patients. These successes have shown the potential of hematopoietic stem cell (HSC) gene therapy, but treatment-related leukemia and benign expansion of gene-modified clones have shifted the attention toward safety. The delayed onset of adverse events in gene therapy clinical trials emphasizes the importance of long-term integration site studies in large animal models. We have addressed safety by characterizing the genomic location of 555 integration sites of the three most commonly used integrating retroviral vectors, that is, gammaretrovirus, lentivirus, and foamy virus, in long-term repopulating cells from dogs. Gammaretroviral integrants showed the most significant frequency of occurrence very close (<2.5 kb) to transcription start sites, but a substantial portion of all three retroviral integrants were within 50 kb. Importantly, gammaretroviral integrants were found more frequently in and near proto-oncogenes, suggesting this retroviral system may be the most prone to adverse gene activation. These data suggest that gammaretroviral vectors may have the highest intrinsic risk, but also emphasize that no vector system can be defined as "safe" based solely on integration profile.
Descriptors: gammaretrovirus genetics, lentivirus genetics, spumavirus genetics, transduction, genetic, virus integration genetics, base sequence, cpg islands, dna primers genetics, dogs, gene therapy adverse effects, gene therapy methods, genetic vectors, models, animal, proto oncogenes, repetitive sequences, nucleic acid, safety.

Beard, B.C. and H.P. Kiem (2009). Canine models of gene-modified hematopoiesis. Methods in Molecular Biology Clifton, N.J. 506: 341-61. ISSN: 1064-3745.
Abstract: Large animal models have played a crucial role in the development of gene therapy protocols. A significant advantage of large animal models over rodent models includes the ability to more easily translate protocols developed in large animals to humans. For gene therapy applications, nonhuman primates and canines have been the main large animal models. Canines have the advantage that there are disease models available, e.g., hemolytic anemia (pyruvate kinase deficiency), leukocyte adhesion deficiency, severe combined immunodeficiency (XSCID), storage diseases, and others. In addition, all three major integrating virus systems, i.e., gammaretrovirus-, HIV-derived lenti- and foamy virus vectors are able to efficiently transduce canine hematopoietic cells. Here we describe protocols developed for efficient transduction of canine hematopoietic repopulating cells.
Descriptors: hematopoiesis genetics, models, animal, antigens, cd34 immunology, cells, cultured, dogs, flow cytometry, gene therapy, genetic vectors, hematopoietic stem cells immunology, retroviridae genetics, transduction, genetic.

Breen, M. (2008). Canine cytogenetics: from band to basepair. Cytogenetic and Genome Research 120(1-2): 50-60. ISSN: 1424-8581.
NAL Call Number: QH431 .C95
Abstract: Humans and dogs have coexisted for thousands of years, during which time we have developed a unique bond, centered on companionship. Along the way, we have developed purebred dog breeds in a manner that has resulted unfortunately in many of them being affected by serious genetic disorders, including cancers. With serendipity and irony the unique genetic architecture of the 21st century genome of Man's best friend may ultimately provide many of the keys to unlock some of nature's most intriguing biological puzzles. Canine cytogenetics has advanced significantly over the past 10 years, spurred on largely by the surge of interest in the dog as a biomedical model for genetic disease and the availability of advanced genomics resources. As such the role of canine cytogenetics has moved rapidly from one that served initially to define the gross genomic organization of the canine genome and provide a reliable means to determine the chromosomal location of individual genes, to one that enabled the assembled sequence of the canine genome to be anchored to the karyotype. Canine cytogenetics now presents the biomedical research community with a means to assist in our search for a greater understanding of how genome architectures altered during speciation and in our search for genes associated with cancers that affect both dogs and humans. The cytogenetics 'toolbox' for the dog is now loaded. This review aims to provide a summary of some of the recent advancements in canine cytogenetics. Copyright 2008 S. Karger AG, Basel.
Descriptors: genetic disorders, cancer, animal models, canine genome, chromosomal location, gene sequence

Casal, M., S. Ryan, J. Rhodes, and J. Scheidt (2003). Immunological aspects of x-linked ectodermal dysplasia: a dog model for the human disease. American Journal of Human Genetics 73(5): 454. ISSN: 0002-9297.
NAL Call Number: QH431.A1A54
Descriptors: molecular genetics, biochemistry and molecular biophysics, X linked ectodermal dysplasia, genetic disease, immune system disease, integumentary system disease, respiratory system disease, genetics, immunology, pathology, transmission, pulmonary disorders, respiratory system disease, respiratory tract infection, infectious disease, cytokine production, immune system defects, immunological aspects, systemic, localized humoral, cellular responses.

Chuah, M.K.L., G. Schiedner, L. Thorrez, B. Brown, M. Johnston, V. Gillijns, S. Hertel, N. Van Rooijen, D. Lillicrap, D. Collen, T. Vandendriessche, and S. Kochanek (2003). Therapeutic factor viii levels and negligible toxicity in mouse and dog models of hemophilia a following gene therapy with high-capacity adenoviral vectors. Blood 101(5): 1734-1743. ISSN: 0006-4971.
NAL Call Number: RB145.A1B57
Abstract: High-capacity adenoviral (HC-Ad) vectors expressing B-domain-deleted human or canine factor VIII from different liver-specific promoters were evaluated for gene therapy of hemophilia A. Intravenous administration of these vectors into hemophilic FVIII-deficient immunodeficient SCID mice (FVIIIKO-SCID) at a dose of 5X109 infectious units (IU) resulted in efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 15 000 mU/mL), correcting the bleeding diathesis. Injection of only 5X107 IU still resulted in therapeutic FVIII levels. In immunocompetent hemophilic FVIII-deficient mice (FVIIIKO), FVIII expression levels peaked at 75 000 mU/mL but declined thereafter because of neutralizing anti-FVIII antibodies and a cellular immune response. Vector administration did not result in thrombocytopenia, anemia, or elevation of the proinflammatory cytokine interleukin-6 (IL-6) and caused no or only transient elevations in serum transaminases. Following transient in vivo depletion of macrophages before gene transfer, significantly higher and stable FVIII expression levels were observed. Injection of only 5X106 HC-Ad vectors after macrophage depletion resulted in long-term therapeutic FVIII levels in the FVIIIKO and FVIIIKO-SCID mice. Intravenous injection of an HC-Ad vector into a hemophilia A dog at a dose of 4.3X109 IU/kg led to transient therapeutic canine FVIII levels that partially corrected whole-blood clotting time. Inhibitory antibodies to canine FVIII could not be detected, and there were no signs of hepatotoxicity or of hematologic abnormalities. These results contribute to a better understanding of the safety and efficacy of HC-Ad vectors and suggest that the therapeutic window of HC-Ad vectors could be improved by minimizing the interaction between HC-Ad vectors and the innate immune system.
Descriptors: blood and lymphatics, transport and circulation, genetics, molecular genetics, biochemistry and molecular biophysics, hemophilia A, blood and lymphatic disease, genetic disease, gene therapy, genetic techniques, laboratory techniques.

Cox, M.L., G.E. Lees, C.E. Kashtan, and K.E. Murphy (2003). Genetic cause of X-linked Alport syndrome in a family of domestic dogs. Mammalian Genome. 14(6): 396-403. ISSN: 0938-8990.
NAL Call Number: QL737.R638M68
Abstract: Alport syndrome is a hereditary disease of type IV (basement membrane) collagens that occurs spontaneously in humans and dogs. In the human, X-linked Alport syndrome (XLAS) is caused by mutations in COL4A5, resulting in absence of type IV collagen alpha 5 chains from the glomerular basement membrane (GBM) of affected individuals. The consequence of this defect is progressive renal failure, for which the only available treatments are dialysis and transplantation. Recent studies support the prospect of gene transfer therapy for Alport syndrome, but further development of required technologies and demonstration of safety and efficacy must be accomplished in a suitable animal model. We previously identified and have propagated a family of mixed-breed dogs with an inherited nephropathy that exhibits the clinical, immunohistochemical, pathological, and ultrastructural features of human XLAS. To identify the causative mutation, COL4A5 cDNAs from normal and affected dogs were sequenced in their entirety. Sequence analyses revealed a 10-bp deletion in exon 9 of affected dogs. This deletion causes a frame-shift that results in a premature stop codon in exon 10. Characterization of the causative mutation was followed by development of an allele-specific test for identification of dogs in this kindred that are destined to develop XLAS.
Descriptors: alleles, amino acid sequences, codons, collagen, complementary DNA, deletions, DNA sequencing, exons, genetic disorders, hereditary diseases, kidney diseases, mutations, nucleotide sequences, renal failure, X chromosome, dogs.

Creevy, K.E., T.R.J. Bauer, L.M. Tuschong, L.J. Embree, L. Colenda, K. Cogan, M.F. Starost, M.E. Haskins, and D.D. Hickstein (2003). Canine leukocyte adhesion deficiency colony for investigation of novel hematopoietic therapies. Veterinary Immunology and Immunopathology 94(1/2): 11-22. ISSN: 0165-2427.
NAL Call Number: SF757.2.V38
Abstract: The genetic immunodeficiency disease canine leukocyte adhesion deficiency (CLAD) was originally described in juvenile Irish Setters with severe, recurrent bacterial infections. CLAD was subsequently shown to result from a mutation in the leukocyte integrin CD18 subunit which prevents leukocyte surface expression of the CD11/CD18 complex. We describe the development of a mixed-breed CLAD colony with clinical features that closely parallel those described in Irish Setters. We demonstrate that the early identification of CLAD heterozygotes and CLAD-affected dogs by a combination of flow cytometry and DNA sequencing allows the CLAD-affected animals to receive life-saving antibiotic therapy. The distinct clinical phenotype in CLAD, the ability to detect CD18 on the leukocyte surface by flow cytometry, and the history of the canine model in marrow transplantation, enable CLAD to serve as an attractive large-animal model for the investigation of novel haematopoietic stem cell and gene therapy strategies.
Descriptors: diagnosis, DNA sequencing, flow cytometry, genetic disorders, heterozygotes, leukocyte disorders, leukocytes.

Ellinwood, N.M., P.S. Henthorn, U. Giger, and M.E. Haskins (2003). Mucopolysaccharidosis type iiib: identification of the causative mutation in the canine model. American Journal of Human Genetics 73(5): 449. ISSN: 0002-9297.
NAL Call Number: QH431.A1A54
Descriptors: enzymology, biochemistry and molecular biophysics, molecular genetics, biochemistry and molecular biophysics, mucopolysaccharidosis type IIIB, connective tissue disease, genetic disease, metabolic disease

Ford, M.M., R. Bragadottir, E. Rakoczy, and K. Narfstrom (2003). Long - term visual behavior correlates with functional electroretinographic assessment after gene transfer in rpe65 - / - dogs. Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA; May 04-08, 2003,
Abstract: Purpose: To assess the long-term effects of subretinal rAAV.RPE65 gene transfer on functional vision in RPE65-/- dogs. Methods: Five dogs homozygous for the RPE65 null mutation and 2 unaffected, visually normal control dogs were included. Affected animals had been treated subretinally with 70 - 100 mul of rAAV.RPE65 gene construct (2 X 1012 particles/ml) in the right eye (OD), and 0 - 100 mul of rAAV.GFP (2 X 1010 transforming units/ml) in the left eye (OS). Treatment for control dogs was 75 - 100 mul of the latter OD and 100 mul of BSS OS. Evaluations from pre-surgical and follow-up bilateral full-field electroretinograms (ERGs) 6 mo post-surgery were obtained. Objective assessment of functional vision was performed 3 and 6 mo post-operatively by counting the number of collisions made by the dogs as they negotiated a maze of obstacles in dim and day light conditions. Vision in individual eyes was also assessed 6 mo post-surgery. Results: Three mo after gene therapy there was a higher number of collisions in dim light than in day light (p<0.008) among affected dogs; a difference not evident 6 mo post-operatively. In day light conditions, no difference was noted between control and affected animals at 3 or 6 mo. In dim light, affected dogs had a higher number of collisions than control dogs at 3 (p<0.004) but not 6 mo post-operatively. Fewer collisions (p<0.1) were noted in day and dim light when only OD was exposed compared with exposure of only OS in the affected dogs. Post-surgery, high intensity scotopic stimuli and 30 Hz flicker ERG amplitudes were elevated (p<0.04) over pre-operative recordings in OD, and elevated over post-operative recordings in OS as well (p<0.032). Further, post-operative single flash photopic b-wave amplitudes for OS were increased over pre-operative values (p<0.05). Conclusions: Progressive improvement in functional binocular and monocular (OD) vision up to 6 mo after gene transfer was found by objective behavioral testing. The long-term visual improvement correlated with a significant increase in post-operative ERG amplitudes, mainly in OD but also with measurable effects in OS.
Descriptors: methods and techniques, molecular genetics, biochemistry and molecular biophysics, sense organs, sensory reception, functional electroretinography, laboratory techniques, gene transfer, genetic techniques, visual behavior.

Ford, M., R. Bragadottir, P.E. Rakoczy, and K. Narfstrom (2003). Gene transfer in the rpe65 null mutation dog: relationship between construct volume, visual behavior and electroretinographic (erg) results. Documenta Ophthalmologica 107(1): 79-86. ISSN: 0012-4486.
Abstract: In vivo gene transfer in a large group of RPE65 null mutation dogs have been recently performed. The present study was aimed at determining, through visual behavioral and electroretinographic (ERG) testing, if there is a volume effect of the gene construct administered. Eleven Beagle-Briard dogs homozygous for the RPE65 null mutation and two unaffected control dogs were included. Affected animals were unilaterally treated with either a high (70-100 mul; N=6) or a low volume (30-60 mul; N=5) of subretinally injected rAAV.RPE65 construct, at the age of 4 months to 2.5 years. Fellow eyes were treated with a subretinal injection of rAAV.GFP or sham operated and used as internal controls. Retinal function was measured pre- and 10-12 weeks post-surgically, using simultaneous bilateral full-field flash ERGs. A significant improvement in all ERG responses studied was identified for the high volume treated group compared to pre-surgical parameters. A significant improvement for the high intensity scotopic a-wave response for the low volume rAAV.RPE65 treated group was also found. Objective and subjective dim and day light visual maze testing, in eight of the affected treated animals, and the two control dogs, revealed better vision in daylight than in dim light for all animals. Vision in dogs treated with the high volume of gene construct was significantly better in day light than in dim light. No significant difference was noted between day and dim light testing for the control group or those animals treated with a low volume of the gene construct. Significantly better vision was noted in the control group when compared with the low volume group under dim light conditions, and the high volume group under day light conditions. No significant difference in functional vision could be identified between the high volume treated animals and control animals in day light conditions. These findings support the hypothesis that functional vision is improved by subretinal rAAV.RPE65 injection in a volume-dependent manner.
Descriptors: methods and techniques, molecular genetics, biochemistry and molecular biophysics, sense organs, sensory reception, electroretinogram, clinical techniques, diagnostic techniques, in vivo gene transfer, genetic techniques, laboratory techniques, gene transfer, visual behavior.

Green, S.L., D.M. Bouley, M.J. Pinter, L.C. Cork, and G.T. Vatassery (2001). Canine motor neuron disease: clinicopathologic features and selected indicators of oxidative stress. Journal of Veterinary Internal Medicine 15(2): 112-119. ISSN: 0891-6640.
NAL Call Number: SF601.J65
Abstract: Hereditary canine spinal muscular atrophy (HCSMA) is an inherited motor neuron disease affecting a kindred of Brittanies. We have examined the clinicopathologic abnormalities in 57 animals with HCSMA, including 43 affected adult dogs and 14 homozygote pups. We also measured selected biochemical indices of oxidative stress: serum vitamin E (alpha-tocopherol) and Se concentrations; serum concentrations of Cu, Zn, Mg, and Fe; and total superoxide dismutase and glutathione peroxidase activities in red blood cells. Dogs with HCSMA had the following abnormalities: regenerative anemia, hypoglobulinemia, hypochloremia, and abnormally high creatine kinase and liver alkaline phosphatase activities. Serum Cu concentration was significantly (P = .01) increased in adult dogs with HCSMA compared to control dogs. Serum vitamin E concentrations tended to be lower in adult dogs with HCSMA compared to controls, and were significantly (P = .01) lower in homozygote pups compared to control pups.
Descriptors: veterinary medicine, medical sciences, neural coordination, hereditary canine spinal muscular atrophy, nervous system disease, motor neuron disease, muscle disease, clinicopathologic features, oxidative stress.

Higgins, M.A., B.R. Berridge, B.J. Mills, A.E. Schultze, H. Gao, G.H. Searfoss, T.K. Baker, and T.P. Ryan (2003). Gene expression analysis of the acute phase response using a canine microarray. Toxicological Sciences 74(2): 470-484. ISSN: 1096-6080.
NAL Call Number: RA1190.F8
Abstract: The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n = 3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.
Descriptors: immune system, chemical coordination and homeostasis, molecular genetics, biochemistry and molecular biophysics, toxicology, affymetrix based oligonucleotide microarray, genetic techniques, laboratory techniques, canine microarray, gene expression analysis, acute phase response, biotransformation, canine genomics, extracellular matrix remodeling, glucose homeostasis, hepatic gene expression, inflammation, toxicogenomics .

Horwitz, M., K.F. Benson, Z. Duan, F.Q. Li, and R.E. Person (2004). Hereditary neutropenia: dogs explain human neutrophil elastase mutations. Trends in Molecular Medicine 10(4): 163-170 ISSN: 1471-4914.
NAL Call Number: QH506
Abstract: Mutations in ELA2 the gene encoding neutrophil elastase (NE), cause the human diseases cyclic neutropenia (CN) and severe congenital neutropenia (SCN). Numerous mutations are known, but their lack of consistent biochemical effect has proven puzzling. The recent finding that mutation of AP3B1, which encodes the P subunit of adaptor protein complex 3 (AP3), is the cause of canine CN suggests a model for the molecular basis of hereditary neutropenias, involving the mistrafficking of NE: AP3 recognizes NE as a cargo protein, and their interaction implies that NE is a transmembrane protein. Computerized algorithms predict two NE transmembrane domains. Most CN mutations fall within predicted transmembrane domains and lead to excessive deposition of NE in granules, whereas SCN mutations usually disrupt the AP3 recognition sequence, resulting in excessive transport to the plasma membrane.
Descriptors: molecular genetics, biochemistry and molecular biophysics, cyclic neutropenia, blood and lymphatic disease, genetics, hereditary neutropenia, blood and lymphatic disease, genetic disease, immune system disease, severe congenital neutropenia, computerized algorithm, mathematical and computer techniques, gene product interactions.

Hunter, L.S., D.J. Sidjanin, G.M. Acland, M. Villagrasa, and G.D. Aguirre (2003). Evaluation of pax6 for mutations causing inherited ocular diseases in canine models. Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA; May 04-08, 2003,
Abstract: Purpose: Normal ocular development is dependent on the transcription factor PAX6. Mutations in PAX6 can result in aniridia, cataract, glaucoma, foveal hypoplasia and optic nerve hypoplasia in man. In mice, mutations in Pax6 cause the Small Eyepheonotype characterized by microphthalmia, abnormal lens and iris development and cataract. The purpose of this study is to evaluate the canine PAX6 gene for mutations causal to inherited ocular disease in canine models. Methods: Nine breeds of dog were identified having inherited forms of ocular disease for which PAX6 is a candidate gene. Two affected dogs from each breed were evaluated and their PAX6 sequence compared to canine wildtype sequence. Genomic DNA was prepared from whole blood and exons were scanned using intronic primers. Since the sequence for intron 4 could not be amplified, exon 4 was sequenced using primers in intron 3 and at the 3' end of exon 4, and exon 5 was sequenced using primers at the 5' end of exon 5 and in intron 5. Similarly, because of a homopolymer in the 3'UTR, exon 13 was evaluated using primers in intron 12 and at the 3' end of exon 13. Results: Thirteen exons plus an alternatively spliced exon (5a) are being examined in nine dog breeds with inherited eye diseases including cataracts, microphthalmia, persistent pupillary membranes, rod-cone dysplasia 2, cone-rod dystrophy 1 and 2, and aniridia. To date, no mutations associated with disease have been identified in the exons so far examined including the non-coding exons 1, 2, and 3. In addition to a previously identified SNP in intron 8, a single nucleotide change was identified in exon 7. This was a Cytosine -> Thymine change which did not result in an amino acid change. Microsatellites have been identified in exons 1 and 2, and in introns 6 and 7 which may facilitate linkage analysis. Conclusions: PAX6 plays an integral role in ocular development and mutations in this transcription factor cause different disease phenotypes. Evaluating canine models may identify novel disease phenotypes associated with PAX6 mutations.
Descriptors: molecular genetics, biochemistry and molecular biophysics, sense organs, sensory reception, aniridia, congenital disease, eye disease, genetics, cataract, cone rod dystrophy 1, cone rod dystrophy 2, inherited ocular disease, genetic disease, genetics, microphthalmia, persistent pupillary membrane, linkage analysis, genetic techniques, laboratory techniques, disease phenotype, ocular development.

Juraschko, K., A. Meyer Lindenberg, I. Nolte, and O. Distl (2003). Analysis of systematic effects on congenital sensorineural deafness in german dalmatian dogs. Veterinary Journal 166(2): 164-169. ISSN: 1090-0233.
NAL Call Number: SF601.V484
Abstract: We have analysed the systematic influences, phenotypic colour markers and the additive genetic variation for congenital sensorineural deafness (CSD) in German Dalmatian dogs in order to help elucidate the importance of phenotypic breed characteristics for genetic differences of CSD. Linear animal models using restricted maximum likelihood methods were employed to estimate variance components. Data were obtained from all three German Dalmatian kennel clubs associated with the German Association for Dog Breeding and Husbandry (VDH). CSD was recorded by standardized protocols for brainstem auditory-evoked response (BAER). The material included 1899 German Dalmatian dogs from 354 litters in 169 different kennels. BAER testing results were from the years 1986 to 1999. Pedigree information was available for up to seven generations. The animal model regarded the fixed effects of sex, coat colour, eye colour, presence of patches, litter size, percentage of examined puppies per litter, kennel club, and inbreeding coefficient. The common environment of the litter and kennel as well as the additive genetic effect of the animal were taken into account as randomly distributed effects. The fixed effects of eye colour, percentage of puppies examined per litter and kennel club were significant in the mixed model analysis. A significant proportion of additive genetic variation could be shown despite corrections for phenotypic colour variants. The heritability estimate for CSD in German Dalmatian dogs was h2 = 0.27 +- 0.07. The additive genetic correlation of CSD with presence of blue eyes was rg = 0.53 +- 0.41 and with presence of patches rg = -0.36 +- 0.24. We concluded that additional genes other than those associated with phenotypic colour markers in German Dalmatian dogs significantly contribute to the occurrence of CSD.
Descriptors: molecular genetics, biochemistry and molecular biophysics, nervous system, neural coordination, sense organs, sensory reception, veterinary medicine, medical sciences, congenital sensorineural deafness, CSD, congenital disease, ear disease, nervous system disease, diagnosis, genetics, therapy, german association for dog breeding and animal husbandry, brainstem auditory evoked response, breeding, genetic variation, phenotypic color markers.

Konety, B.-R., T.-S. Griffith, S.-J. Sirintrapun, J.-M. Rippentrop, A.-H. Stolpen, B.-R. De-Young, T.-L. Ratliff, and R.-D. Williams (2003). Effect of intra-prostatic injection of adenovirus-trail in vivo in the dog prostate. Journal of Urology 169(4 Supplement): 212. ISSN: 0022-5347.
NAL Call Number: 448.8 J8234
Descriptors: methods and techniques, molecular genetics, biochemistry and molecular biophysics, reproductive system, reproduction , h and e staining, hematoxylin eosin staining, histology and cytology techniques, laboratory techniques, intra prostatic injection, clinical techniques, pelvic computed tomography, imaging and microscopy techniques, radical prostatectomy, therapeutic and prophylactic techniques, periprostatic inflammation.

Schneider, M.R., H. Adler, J. Braun, B. Kienzle, E. Wolf, and H.J. Kolb (2007). Canine embryo-derived stem cells--toward clinically relevant animal models for evaluating efficacy and safety of cell therapies. Stem Cells Dayton, Ohio 25(7): 1850-1. ISSN: 1066-5099.
Descriptors: embryo, mammalian cytology, embryonic stem cells cytology, models, animal, tissue therapy adverse effects, antigens, cd34 genetics, antigens, cd34 metabolism, cell differentiation, cell separation, dogs, gata2 transcription factor genetics, gata2 transcription factor metabolism, gene expression regulation, rna, messenger genetics, rna, messenger metabolism, treatment outcome.

Shelton, G.D., M. Podell, L. Poncelet, S. Schatzberg, E. Patterson, H.C. Powell, and A.P. Mizisin (2003). Inherited polyneuropathy in leonberger dogs: a mixed or intermediate form of charcot-marie-tooth disease? Muscle and Nerve 27(4): 471-477. ISSN: 0148-639X.
Abstract: A spontaneous distal, symmetrical polyneuropathy in related Leonberger dogs with onset between 1 to 9 years of age was characterized clinically, electrophysiologically, histologically, and morphometrically. Exercise intolerance and weakness was associated with a high-steppage pelvic-limb gait, a loss or change in the pitch of the bark, and dyspnea. Neurological examination revealed marked atrophy of the distal limb muscles, depressed spinal and cranial nerve reflexes, and weak or absent movement of the laryngeal and pharyngeal muscles. Electrophysiological evaluation was consistent with denervation and was characterized by loss or marked attenuation of compound muscle action potentials and slowed motor nerve conduction velocity. Muscle biopsy specimens showed neurogenic atrophy. Chronic nerve fiber loss associated with decreased myelinated fiber density and a shift of the axonal size-frequency distribution toward smaller fibers was the predominant finding in peripheral nerve specimens. Pedigree analysis of a large multigenerational family, including nine sibships with at least one affected individual, suggested X-linked inheritance. Mutational and linkage analysis of this family may aid in identification of the chromosomal loci and gene responsible for this inherited axonal neuropathy. Further characterization of this inherited axonal neuropathy may establish the Leonberger dog as a spontaneous animal model of inherited axonal neuropathy and possibly lead to the discovery of a new gene or genes associated with axonal variants.
Descriptors: molecular genetics, biochemistry and molecular biophysics, nervous system, neural coordination, Charcot Marie tooth disease, congenital disease, genetic disease, muscle disease, nervous system disease, chronic nerve fiber loss, distal limb muscle atrophy, dyspnea, respiratory system disease, exercise intolerance, inherited polyneuropathy, genetic disease, muscle weakness, neurogenic atrophy, cranial nerve reflex, motor nerve conduction velocity, myelinated fiber density, spinal nerve reflex.

Shin, D. and C. Park (2004). N-terminal extension of canine glutamine synthetase created by splicing alters its enzymatic property. Journal of Biological Chemistry 279(2): 1184-1190. ISSN: 0021-9258.
NAL Call Number: 381 J824
Abstract: It was found that an extra exon exists in the first intron of glutamine synthetase gene, generated by means of alternative splicing (Shin, D., Park, S., and Park, C. (2003) Biochem. J. 374, 175-184). Inclusion of this exon decreased the translation of glutamine synthetase (GS) in human, dog, and mouse. When translated in vitro with the canine GS transcript containing the exon, we obtained two different species of GS enzymes. Besides the known 45-kDa protein, the extended form of GS was identified with additional 40 amino acids on its N-terminal end. An upstream ATG in the extra exon served as a translation initiator for the long form of GS. When the long transcript was translated in vivo in animal cells, only the long GS was expressed. On the other hand, the long GS is less predominant relative to the short one in canine tissues including brain and liver. Subcellular fractionation of canine brain revealed that the long GS is present in all cellular compartments as is the short one, which is consistent with fluorescence microscopy data obtained with green fluorescent protein fused to GS. The short (SGS) and long (LGS) forms of canine GS were purified in Escherichia coli and shown to have similar Km values for L-glutamate and hydroxylamine. However, the Km values for ATP were slightly altered, 1.3 and 1.9 mM for the short and long GSs, respectively. The Kis for L-methionine-S-sulfoximine (MSOX), a highly potent ATP-dependent inactivator of GS, were considerably different such that the values are 0.067 and 0.124 mM for the short and long forms, respectively. When the intrinsic fluorescences of tryptophans were monitored upon bindings of chloride and metal ions without any effect on the oligomeric state, the pattern of quenching in LGS was significantly different from that of SGS. Taken together, the N-terminal extension in the long isoform of GS induces a conformational change of core enzyme, leading to a change in affinity to its substrates as well as in the effector-induced conformational alterations.
Descriptors: enzymology, biochemistry and molecular biophysics, genetics, metabolism .

Shin, D., S. Park, and C. Park (2003). A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene. Biochemical Journal 374(1): 175-184. ISSN: 0264-6021.
NAL Call Number: QP501.B64
Abstract: The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5'-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase-PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1'. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5'-untranslated region (UTR) containing the exon 1'. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin-Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5'-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5'-UTRs, demonstrating that the long form with extra 5'-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5'-UTRs were less efficient, showing 6-8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1', the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5'-UTR is, to some extent, due to an abortive translation from the upstream ATG.
Descriptors: molecular genetics, biochemistry and molecular biophysics, 5' circular rapid amplification of cdna ends, 5' crace, genetic techniques, laboratory techniques, reverse transcriptase pcr, rt pcr, genetic techniques, laboratory techniques, alternative splicing, gene expression.

Vassilopoulos, G. and A. Rethwilm (2008). The usefulness of a perfect parasite. Gene Therapy 15(19): 1299-1301.
Descriptors: gene therapy methods, leukocyte adhesion deficiency syndrome therapy, dogs, genetic vectors administration and dosage, genetic vectors genetics, models, animal, spumavirus genetics, transduction, genetic methods.

Yuasa, K., M. Yoshimura, N. Urasawa, S. Ohshima, J.M. Howell, A. Nakamura, T. Hijikata, Y. Miyagoe Suzuki, and S. Takeda (2007). Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Therapy 14(17): 1249-60. ISSN: 0969-7128.
Abstract: Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding beta-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of beta-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.
Descriptors: dependovirus genetics, gene therapy adverse effects, genetic vectors adverse effects, muscle, skeletal immunology, muscular dystrophy, animal therapy, muscular dystrophy, duchenne therapy, base sequence, calmodulin genetics, cyclosporine administration and dosage, dogs, dystrophin genetics, dystrophin metabolism, gene therapy methods, genetic engineering, genetic vectors genetics, immunosuppressive agents administration and dosage, injections, intramuscular, interferon gamma immunology, lymphocyte activation, mice, mice, inbred c57bl, models, animal, molecular sequence data, muscle fibers, skeletal immunology, muscle fibers, skeletal virology, muscular dystrophy, animal immunology, muscular dystrophy, duchenne immunology, parvoviridae infections immunology, rna, messenger analysis, reverse transcriptase polymerase chain reaction, species specificity, t lymphocytes, cytotoxic immunology, transduction, genetic methods, transgenes, beta galactosidase genetics.

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