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United States Department of Agriculture

Agricultural Research Service

Research Project: IMMUNOLOGIC AND PHARMACOLOGICAL INTERVENTIONS OF VECTOR-BORNE BABESIOSIS

Location: Animal Diseases Research

Title: Transfection systems for Babesia bovis: a review of methods for the transient and stable expression of exogenous genes

Authors
item Suarez, Carlos
item Mcelwain, Terry -

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 3, 2009
Publication Date: February 10, 2010
Repository URL: http://ddr.nal.usda.gov/dspace/bitstream/10113/40788/1/IND44319961.pdf
Citation: Suarez, C.E., Mcelwain, T.F. 2010. Transfection systems for Babesia bovis: a review of methods for the transient and stable expression of exogenous genes. Veterinary Parasitology. 167(2-4):205-215.

Interpretive Summary: With the recently sequenced B. bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.

Technical Abstract: With the recently sequenced B. bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3’ region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1' promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovis ef-1' locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.

Last Modified: 4/18/2014
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