TY - JOUR
T1 - Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni
AU - Rinaldi, Gabriel
AU - Yan, Hongbin
AU - Nacif-Pimenta, Rafael
AU - Matchimakul, Pitchaya
AU - Bridger, Joanna
AU - Mann, Victoria H.
AU - Smout, Michael J.
AU - Brindley, Paul J.
AU - Knight, Matty
N1 - Funding Information:
We thank Dr. Irene Riz, who generously provided the plasmid piLenti-RNAi-GFP and the HEK293T cells. These studies were supported by awards 1R21AI109532-01A1 (G.R., V.H.M., P.J.B.) and 1R01AI072773 (P.J.B., V.H.M., G.R.) from the National Institute of Allergy & Infectious Diseases , National Institutes of Health , USA.
Publisher Copyright:
© 2015 Australian Society for Parasitology Inc.
PY - 2015/7/1
Y1 - 2015/7/1
N2 - 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.
AB - 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.
KW - Antibiotic selection
KW - Biomphalaria glabrata
KW - Cytometrics
KW - Genetic transformation
KW - Molluscan embryonic cell line (Bge)
KW - XCELLigence real time cellular analysis (RTCA)
KW - Cell Line
KW - Biomphalaria/drug effects
KW - Puromycin/pharmacology
KW - Anti-Bacterial Agents/pharmacology
KW - Animals
KW - Schistosoma mansoni/physiology
KW - Xenobiotics/pharmacology
KW - Host-Parasite Interactions
UR - http://www.scopus.com/inward/record.url?scp=84929656007&partnerID=8YFLogxK
U2 - 10.1016/j.ijpara.2015.02.012
DO - 10.1016/j.ijpara.2015.02.012
M3 - Article
C2 - 25907768
AN - SCOPUS:84929656007
SN - 0020-7519
VL - 45
SP - 527
EP - 535
JO - International Journal for Parasitology
JF - International Journal for Parasitology
IS - 8
ER -