Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.
Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.
Gaji
Rajshekhar Y
RY
108 M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA.
Zhang
Deqing
D
Breathnach
Cormac C
CC
Vaishnava
Shipra
S
Striepen
Boris
B
Howe
Daniel K
DK
D024506:5' Flanking Region; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D000981:Antiprotozoal Agents / Q000494:pharmacology; D015723:Gene Library; D018503:Genome, Protozoan; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D012522:Sarcocystis / Q000187:drug effects / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
D000953:Antigens, Protozoan; D000981:Antiprotozoal Agents; D008164:Luminescent Proteins; D015800:Protozoan Proteins; C097089:p31 protein, Sarcocystis; D001616:beta-Galactosidase; D011739:Pyrimethamine