Feline Stem Cell Factor: Isolation and Characterisation of Biological Activity

Dunham, Stephen P (1997) Feline Stem Cell Factor: Isolation and Characterisation of Biological Activity. PhD thesis, University of Glasgow.

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Cytokines are small proteins produced by many tissue types and have wide ranging effects on the haemopoietic and immune systems. The cloning of human cytokines has facilitated the production of recombinant cytokines in quantities sufficient to enable detailed study of their biological properties. An understanding of the biological effects of these cytokines has led to their introduction as novel therapeutic agents with widespread potential uses, including the treatment of cancer, cytopenias and viral infections. The use of heterologous cytokines in domestic species has been of only limited success, in part due to the variable degree of interspecies conservation. In order to fully realise the potential for cytokines as therapeutic agents and to facilitate further studies of the role of cytokines in diseases of domestic species, the isolation of species specific cytokines is desirable. This thesis describes the approach used to isolate and clone feline stem cell factor (fSCF) and subsequently express the recombinant protein and characterise its biological properties. Stem cell factor is the ligand for the tyrosine kinase receptor encoded by the c-kit gene. It has wide ranging actions on cells of the haemopoietic, reproductive and nervous systems and melanocytes, in particular promoting the survival and development of primitive cells. cDNA clones encoding two isoforms of fSCF were isolated using RT-PCR and their sequences determined. The cDNAs encode a predicted full length fSCF protein of 274 amino-acids and a shorter isoform of 246 amino acids. Feline SCF shows a high degree of homology to the SCFs of other species at both the nucleic acid and protein level. Feline SCF was expressed as a soluble protein using the glutathione S-transferase fusion protein system and purified by affinity, anion exchange and gel filtration chromatography. Murine MC/9 and human TF-1 cells were used to assay fSCF biological activity. The recombinant protein supported the growth of feline granulocyte-macrophage colony forming cells in vitro and in combination with feline phytohaemagglutinin lymphocyte conditioned medium increased colony numbers and sizes were seen. Administration of the recombinant protein to cats produced increases in circulating colony forming cells, induced extramedullary haemopoiesis in the spleens of treated cats and led to increased mast cell numbers at the site of injection. In order to enable assessment of the effects of frSCF upon primitive haemopoietic cells, the production of polyclonal antiserum to CD34 (a transmembrane glycoprotein expressed predominantly on primitive haemopoietic cells) was attempted. Rabbits were used to raise antisera to conserved intracellular epitopes of the CD34 molecule by inoculation with immunogenic peptides. This was of limited success; whilst the antisera recognised the synthetic peptides against which they had been raised, they showed poor affinity for the native protein. These studies provide the basis for further investigations of the potential of this cytokine in the treatment of feline disease, particularly cytopenias associated with neoplasia, chemotherapy or viral disease (e.g. FeLV, FIV) and in the development of peripheral stem cell transplantation. The ability of fSCF to synergise with other cytokines in vitro suggests that it may be combined with other haemopoietic cytokines in vivo to provide more potent haemopoietic stimulation. Furthermore, the recombinant cytokine may be usefully employed to support in vitro growth of haemopoietic cells in this species and so facilitate their study.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Onions
Keywords: Veterinary science
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75314
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:14
Last Modified: 19 Nov 2019 21:14
URI: https://theses.gla.ac.uk/id/eprint/75314

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