This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Members of the phylum Microsporidia are single-celled obligate intracellular protists that infect vertebrate and invertebrate hosts. Fourteen species of microsporidia have been identified as causes of opportunistic and emerging infections in humans and have been associated with persistent diarrhea and systemic disease. Studies on gene function in the microsporidia have been limited by the lack of genetic manipulation techniques, in part due to the presence of a chitinous wall that surrounds the mature infectious spore stage. To begin to address this issue, electroporation methods were employed to determine if a fluorescein-labeled dsRNA oligonucleotide (BLOCK-iT;Invitrogen) could be introduced into the spores of the human microsporidian species, Vittaforma corneae. Organisms were suspended in various electroporation buffers and subjected to multiple exponential decay and square wave electric pulses in the presence of the FITC-dsRNA. The conditions that resulted in optimal uptake of the FITC-dsRNA, as assessed by confocal microscopy, were electroporation of V. corneae in a 4 mm gap cuvette suspended in electroporation buffer (5 mM CaCl2, 10 mM NaCl, 8.7% glycerol, 0.2 M sucrose, 10 mM HEPES, pH 6.8) and exposed to 2 pulses of 2kV/cm for 80 ?sec each using the ECM 830 electroporation unit (BTX). Efficiency of the FITC-oligonucleotide transfection was 19.54% (+ 1.05) and viability, as measured by a limiting-dilution infectivity assay, was reduced by approximately 50%. These results require further optimization to increase transfection efficiency, but open the door to the application of electroporation for studies on gene function and gene manipulation by, for example, RNA interference, in the microsporidia.
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