title: Integration of a transfected gene into the genome of Babesia bovis occurs by legitimate homologous recombination mechanisms.
abstract: This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasite's two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques.
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, United States; Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA 99164-6630, United States. Electronic address: ces@vetmed.wsu.edu.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
authors.affiliation: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA 99164-6630, United States.
authors.forename: Wendell C
authors.initials: WC
authors.lastname: Johnson
authors.affiliation: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA 99164-6630, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Herndon
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, United States.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Laughery
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, United States.
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
mesh_terms: D000818:Animals; D016793:Babesia bovis / Q000235:genetics* / Q000254:growth & development; D001404:Babesiosis / Q000469:parasitology*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D002999:Clone Cells; D004912:Erythrocytes / Q000469:parasitology; D055786:Gene Knockout Techniques; D017125:Genes, Protozoan / Q000235:genetics*; D049452:Green Fluorescent Proteins; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins
keywords: Babesia bovis; Homologous recombination; Merozoites; Transfection