TY - JOUR
T1 - Molecular genetic transfection of the coccidian parasite Sarcocystis neurona
AU - Gaji, Rajshekhar Y.
AU - Zhang, Deqing
AU - Breathnach, Cormac C.
AU - Vaishnava, Shipra
AU - Striepen, Boris
AU - Howe, Daniel K.
N1 - Funding Information:
We gratefully acknowledge the technical assistance of Dr. David Horohov. This research was supported by grants from the Amerman Family Foundation and Fort Dodge Animal Health (to DKH) and grants from NIH-NIAID (to BS). R. Gaji is supported by a Paul Mellon Graduate Student Fellowship in Equine Veterinary Science. Published as Kentucky Agricultural Experiment Station Article No. 06-14-020.
PY - 2006/11
Y1 - 2006/11
N2 - 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 β-galactosidase (β-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 β-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.
AB - 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 β-galactosidase (β-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 β-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.
KW - Apicomplexa
KW - Coccidia
KW - Fluorescence activated cell sorting
KW - Sarcocystis neurona
KW - Stable transformation
KW - Transfection
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U2 - 10.1016/j.molbiopara.2006.06.003
DO - 10.1016/j.molbiopara.2006.06.003
M3 - Article
C2 - 16844242
AN - SCOPUS:33748308699
SN - 0166-6851
VL - 150
SP - 1
EP - 9
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -