Stable expression of red fluorescent protein-blasticidin deaminase fusion gene (rfp-bsd) as a selectable marker for DNA transfection in Babesia ovata

• Published in: Journal of Veterinary Medical Science 2024, Vol.86(7), pp.744-747
• Main Authors: ARAYASKUL, Nada, ASADA, Masahito, FATHI, Atefeh, ARIEFTA, Nanang R, KOMATSU, Kota, SUGANUMA, Keisuke, INOUE, Noboru, KAWAZU, Shin-ichiro
• Format: Article
• Language: English
• Subjects: Actin; Animals; Babesia - genetics; Babesia ovata; blasticidin deaminase; Blasticidin S; Deoxyribonucleic acid; DNA; Electroporation; Fluorescence microscopy; Fusion protein; Gene fusion; Immunofluorescence; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Parasitology; Plasmids - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Red Fluorescent Protein; selectable marker; Transfection
• ISSN: 0916-7250; 1347-7439; 1347-7439
• DOI: 10.1292/jvms.24-0111

The red fluorescent protein (rfp)-blasticidin deaminase (bsd) fusion gene was transfected into Babesia ovata by electroporation with the plasmid DNA and selected with 15 μg/mL of blasticidin S under the in vitro culture condition. The transfected parasite with episomal DNA was selected and cultured for further analysis based on the presence of the rfp-bsd fusion gene by PCR and expression of the fusion protein by immunofluorescence antibody test under fluorescence microscopy for 2 months after the transfection. The results are the first, to our knowledge, to demonstrate the expression and stability of the episomal rfp-bsd fusion gene under the control of actin promoter as a selectable marker for the transfection system in B. ovata.