title: Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.
abstract: The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transformation, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25°C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (<48 h in 5 μg/ml) facilitated use of this antibiotic, together with the cognate resistance gene (puromycin N-acetyl-transferase) for selection of Bge cells transformed with the PAC gene (puroR). Bge cells transfected with a plasmid encoding puroR were partially rescued when cultured in the presence of 5 μg/ml of puromycin. These findings pave the way for the development of functional genomic tools applied to the host-parasite interaction during schistosomiasis and neglected tropical trematodiases at large.
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA; State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA; Laboratorio de Laboratório de Esquistossomose, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz - FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Nacif-Pimenta
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA; Biomedical Sciences Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand; WHO Collaborating Centre for Research and Control of Opisthorchiasis (Southeast Asian Liver Fluke Disease), Tropical Disease Research Laboratory, Department of Pathology, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Pitchaya
authors.initials: P
authors.lastname: Matchimakul
authors.affiliation: Brunel University London, Kingston Road, Uxbridge, UK.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bridger
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA.
authors.forename: Victoria H
authors.initials: VH
authors.lastname: Mann
authors.affiliation: Australian Institute of Tropical Health and Medicine, Queensland Tropical Health Alliance, James Cook University, McGregor Rd, Smithfield, Cairns, Queensland 4878, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smout
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, Research Center for the Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA. Electronic address: matty_knight@email.gwu.edu.
authors.forename: Matty
authors.initials: M
authors.lastname: Knight
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001700:Biomphalaria / Q000187:drug effects / Q000196:embryology / Q000235:genetics* / Q000469:parasitology; D002460:Cell Line; D006790:Host-Parasite Interactions; D011691:Puromycin / Q000494:pharmacology; D012550:Schistosoma mansoni / Q000502:physiology*; D015262:Xenobiotics / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D015262:Xenobiotics; D011691:Puromycin
keywords: Antibiotic selection; Biomphalaria glabrata; Cytometrics; Genetic transformation; Molluscan embryonic cell line (Bge); xCELLigence real time cellular analysis (RTCA)