In vitro culture and genetic modification of Babesia gibsoni

Babesia gibsoni, a unicellular eukaryotic parasite causing babesiosis in dog, is primarily transmitted through tick feeding. The intraerythrocytic stage, during which the parasite reproduce within the host's red blood cells, is a vital part of Babesia's life cycle. Continuous in vitro culture B. gibsoni provides an opportunity to study its biological processes. The establishment and development of gene editing systems for Babesia offer a powerful tool to investigate the functions of important genes in specific biological processes. This protocol expands on the existing techniques for in vitro culture and genes editing of B. gibsoni. Specifically, we describe a continuous in vitro culture method employing VP-SFM as a base medium, supplemented with Albumax I and small amount of canine serum (2.5 %), This method, designed for long-term culture, achieving high parasitemia and facilitates subclone culture. By employing homology-dependent repair pathways, the gene editing method utilizing introducing homologous fragments and electroporation can effectively manipulate the genetic of B. gibsoni. This protocol would contribute to the reproducibility of experiments and the overall reliability of research findings.