In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L(4) were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L(4) and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Molecular cloning of the cDNA of canine homeodomain-interacting protein kinase 2

The research of p53 is being conducted to find the mechanisms of tumorigenesis and to treat various cancers. Homeodomain-interacting protein kinase2 (HIPK2) is an important factor to regulate p53 and to increase the stability of p53. Activation of HIPK2 leads to the selective phosphorylation of p53, resulting in growth arrest and the enhancement of apoptosis. In this study, the canine HIPK2 cDNA fragments were obtained, and their overlapping regions were aligned to give a total sequence of 3489 bp. The canine HIPK2 cDNA (GenBank accession number; AY800385) shares 93% and 90% sequence identity with those of human and mouse HIPK2, respectively. The canine HIPK2 cDNA contains an open reading frame encoding 1163 amino acid residues and the predicted amino acid sequence has 98% and 96% identity with those of human and mouse, respectively. The deduced amino acid sequence of canine HIPK2 has also all domains' sites compared with human and mouse HIPK2. Therefore, these structural similarities suggested that the canine HIPK2 shares the basic biological functions that HIPK2 exhibit in other species.

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Cloning of canine GM-CSF and SCF genes

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.