Doctor of Philosophy (PhD)
Biomedical and Veterinary Medical Sciences - Veterinary Clinical Sciences
Ehrlichia canis is the etiologic agent of “tropical canine pancytopenia”, or canine ehrlichiosis. The impetus for this research was to overcome the lack of any reliable means of elucidating the genetic profiles of these illusive and historically difficult to manipulate organisms. The use of a broad-host range plasmid greatly facilitated the determination of an electro-transformation protocol. The transforming plasmid possesses a chloramphenicol antibiotic resistance gene marker (chloramphenicol acetyltransferase [CAT] gene), and a visual reporter gene marker, the Green Fluorescent Protein (GFP) gene. With primer sets designed to specifically amplify these two plasmid encoded gene markers, thus verifying the presence of the transforming plasmid, and an additional primer set designed to specifically amplify the p28 gene of E. canis, a multi-copied and species specific genome encoded epitope, PCR verification of electro-transformed E. canis was made possible. Once this PCR protocol was established and accompanied by gel electrophoresis and amplification product sequencing, the presence of electro-transformed E. canis could also be verified by demonstrable antibiotic resistance in tissue culture, as well as fluorescent, light and transmission electron microscopy. Western blot was also performed to verify the expression of GFP in tissue culture by the electro-transformed E. canis.
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Hull, Langston Dolphus, "Plasmid-Mediated Expression of Foreign Genes in Ehrlichia Canis" (2002). LSU Doctoral Dissertations. 2481.