title: Efficient method of cloning the obligate intracellular bacterium Coxiella burnetii.
abstract: Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, 903 S. 4th St., Hamilton, MT 59840, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.forename: Dale
authors.initials: D
authors.lastname: Howe
authors.forename: Diane C
authors.initials: DC
authors.lastname: Cockrell
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002522:Chlorocebus aethiops; D002999:Clone Cells; D016997:Coxiella burnetii / Q000235:genetics / Q000254:growth & development*; D017931:DNA Primers / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D008846:Micromanipulation; D016133:Polymerase Chain Reaction; D014617:Vacuoles / Q000382:microbiology*; D014709:Vero Cells
chemical_list: D017931:DNA Primers