Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker.
Cryptosporidium parvum is a protozoan parasite that infects a variety of mammals. The parasite has been shown to harbor a dsRNA virus (CPV) and in the present study, we have developed a CPV transient transfection system for this parasite by using green fluorescent protein (GFP) to replace the partial gene encoding region of the larger dsRNA (CPV-L) and the smaller dsRNA (CPV-S) virus. Two viral RNA-mediated transfection vectors: pCPVL-GFP and pCPVS-GFP were successfully constructed and both in vitro transcripts were electroporated into oocysts and sporozoites. Transient expression of GFP was detected in C. parvum oocysts and excysted sporozoites by fluorescence microscopy and by RT-PCR detection of GFP mRNA and antisense RNA in transfected C. parvum oocysts. Our study provides a new approach for studying gene expression and regulation in C. parvum and will hopefully lead to the construction of a stable CPV transfection system in the future.
Li
Wei
W
College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xian Road, Changchun 130062, China.
Zhang
Nan
N
Liang
Xiaoying
X
Li
Jianhua
J
Gong
Pengtao
P
Yu
Xinyou
X
Ma
Guangpeng
G
Ryan
U M
UM
Zhang
Xichen
X
D000818:Animals; D002417:Cattle; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000302:isolation & purification; D018274:Electroporation; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008967:Molecular Biology / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
D012330:RNA, Double-Stranded; D049452:Green Fluorescent Proteins