Rinaldi Gabriel, Yan Hongbin, Nacif-Pimenta Rafael, Matchimakul Pitchaya, Bridger Joanna, Mann Victoria H, Smout Michael J, Brindley Paul J, Knight Matty
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.
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.
Int J Parasitol. 2015 Jul;45(8):527-35. doi: 10.1016/j.ijpara.2015.02.012. Epub 2015 Apr 20.
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.
源自光滑双脐螺胚胎的无脊椎动物细胞系Bge，至今仍是软体动物门任何物种中唯一已建立的细胞系。自1976年由埃德尔·汉森建立以来，很少有研究关注其细胞计量学、生长特性或对外源生物的敏感性分析。据报道，Bge细胞的增殖和维持具有挑战性。因此，尽管该细胞系是一项值得关注的资源，但尚未得到广泛研究。随着对功能基因组学（包括基因转化）兴趣的增加，为了阐明负责血吸虫病传播的蜗牛中间宿主的分子层面，并旨在提高这种软体动物细胞系的维持便利性，我们采用xCELLigene实时方法来研究Bge细胞。三种Bge分离株（称为CB、SL和UK）的倍增时间比哺乳动物细胞系更长——在25°C下添加7%胎牛血清的完全Bge培养基中，倍增时间超过40小时，接种40000个细胞时，范围从约42小时到约157小时。为了评估细胞进行基因转化的潜力，探索了抗生素筛选。Bge细胞对来自白色链霉菌的氨基核苷抗生素嘌呤霉素敏感，浓度范围为5μg/ml至200ng/ml，半最大抑制浓度（IC₅₀）约为1.91μg/ml。对嘌呤霉素的敏感性以及相对较快的杀伤时间（在5μg/ml中<48小时）便于使用这种抗生素，连同同源抗性基因（嘌呤霉素N - 乙酰转移酶）用于筛选用PAC基因（puroR）转化的Bge细胞。当在5μg/ml嘌呤霉素存在下培养时，用编码puroR的质粒转染的Bge细胞得到部分挽救。这些发现为开发应用于血吸虫病以及更广泛的被忽视热带吸虫病中宿主 - 寄生虫相互作用的功能基因组工具铺平了道路。
