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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: Optimizacion of Babesia bovis transfection methods

Authors
item SUAREZ, CARLOS
item Laughery, J - WSU
item Gonzalez, MA - WSU
item Mcelwain, T - WSU

Submitted to: Parassitologia
Publication Type: Monograph
Publication Acceptance Date: April 15, 2007
Publication Date: May 1, 2007
Citation: Suarez, C.E., Laughery, J., Gonzalez, M.G., Mcelwain, T. 2007. Optimizacion of Babesia bovis transfection methods. Parassitologia. 49(Suppl. 1):67-70.

Interpretive Summary: The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa).The results indicates that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 micrograms range) whereas the inverse is true for transfection of larger quantities (100 micrograms range). This information will facilitate further development of efficient stable transfection systems.

Technical Abstract: The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa) A comparative study among four different transfection methods: transfection of infected erythrocytes and purified merozoites with 2 or 100 micrograms of plasmid, using electroporation (BioRad GenePulser II) or nucleofection (Amaxa) indicates that electroporation of infected erythrocytes with 100 micrograms of plasmid or nucleofection with 2 micrograms of plasmid are the most efficient ways to transfect B. bovis parasites. The data also indicates that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 micrograms range), whereas the inverse is true for transfection of larger quantities (100 micrograms range). This information will facilitate further development of efficient stable transfection systems.

Last Modified: 8/27/2014
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