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Title: Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites

Author
item Suarez, Carlos
item MCELWAIN, TERRY - WSU

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2008
Publication Date: 2/1/2009
Citation: Suarez, C.E., Mcelwain, T. 2009. Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites. International Journal for Parasitology. 39(3):289-297.

Interpretive Summary: Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, we describe integration and expression of foreign genes in Babesia bovis for the first time. The transfection system uses a plasmid encoding the fluorescent protein gfp fused to blasticidin deaminase, an enzyme that can confer resistance to the drug blasticidin, which has an inhibitory effect for B. bovis. The plasmid was transferred to B. bovis using previously developed electroporation technology and selected with blasticidin in in vitro cultures. Stable transformants emerged from the blasticidin-selected cultures, and one parasite transfected cell line was analyzed in further detail. The stable B. bovis transformant was able to express the foreign gene for at least 9 months in in vitro culture. Integration of the foreign gfp-bsd gene into the genome was demonstrated by Southern blot hybridizations and sequencing of PCR products. Expression of the gfp-bsd fusion protein was demonstrated by fluorescence microscopy, transcriptional analysis and immunoblots. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease, and immunity of B. bovis, one of the 3 major tick borne diseases that limit global livestock production.

Technical Abstract: Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, a plasmid was designed to target integration of a gfp-bsd gene into the B. bovis ef-1' locus. B. bovis infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids, and selected in cultures with varying amounts of blasticidin 24 hours after electroporation. Several blasticidin resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence under fluorescence microscopy in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by RT-PCR, Western blot analysis, and fluorescence microscopy for longer than nine months after electroporation. No plasmid or episomal DNA could be detected by PCR in this line, and plasmid recovery in E. coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1' locus in the transfected parasite line. Overall the results demonstrate for the first time chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease, and immunity of B. bovis, one of the 3 major tickborne diseases that limit global livestock production.