Original Article
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World J Stem Cells. Dec 26, 2010; 2(6): 127-140
Published online Dec 26, 2010. doi: 10.4252/wjsc.v2.i6.127
Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines
Solomon Mamo, Julianna Kobolak, Istvan Borbíró, Tamás Bíró, Istvan Bock, Andras Dinnyes
Solomon Mamo, Julianna Kobolak, Andras Dinnyes, Genetic Reprogramming Group, Agricultural Biotechnology Center, Szent-Gyorgyi Albert ut. 4, H-2100 Gödöllő, Hungary
Solomon Mamo, Lyons Research Farm, University College Dublin, Newcastle, Co Dublin, Ireland
Istvan Borbíró, Tamás Bíró, Department of Physiology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary; Abiol Ltd., Nagyerdei krt. 98, H-4032 Debrecen, Hungary
Istvan Bock, BioTalentum Ltd., Aulich l. 26, Gödöllő, Hungary
Andras Dinnyes, Molecular Animal Biotechnology Laboratory, Szent Istvan University, Páter K. u. 1, H-2103 Gödöllő, Hungary
Author contributions: Mamo S participated in the experimental design, coordinated molecular biology activities, performed real time PCR and microarray analysis, interpretation of the data and was the primary author of the manuscript; Kobolak J conceived the experiment, performed the stem cell culture, Southern blot, and participated in manuscript preparation; Borbíró I performed the Ca2+ assay measurement; Bíró T performed Ca2+ assay data analysis and participated in manuscript preparation; Bock I performed the real time PCR experiment and data analysis for the various Ca2+ receptors; Dinnyes A supervised the study design, execution, analysis, and approved the final version.
Supported by EU FP6 (TEAMOHOLIC “MEXT-CT-2003-509582”, “MEDRAT” LSHG-CT-2006-518240, “CLONET” MRTN-CT-2006-035468), EU FP7 (“PartnErS” PIAPGA-2008-218205; “Plurisys” HEALTH-F4-2009-223485; “RESOLVE” FP7-HEALTH-F4-2008-202047), NKFP_07_1-ES2HEART-HU (OM-00202-2007), Hungarian-Chinese NKTH TET, No. CN-56/2007 and “Plurabit” NKTH TET-09-1-2010-0007
Correspondence to: Andras Dinnyes, PhD, DSc, Professor, Molecular Animal Biotechnology Laboratory, Szent Istvan University, Páter K. u. 1, H-2103 Gödöllő, Hungary. andrasdinnyes@yahoo.com
Telephone: +36-20-5109632 Fax: +36-28-526151
Received: March 24, 2010
Revised: October 5, 2010
Accepted: October 12, 2010
Published online: December 26, 2010

AIM: To compare gene targeting efficiencies, expression profiles, and Ca2+ handling potentials in two widely used mouse embryonic stem cell lines.

METHODS: The two widely used mouse embryonic stem cell lines, R1 and HM-1, were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers, following standard culture procedures. Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers. Moreover, cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct, following the BioRad and Amaxa transfection procedures. Subsequently, the cells were cultured and neomycin-resistant cells were picked after 13 d of selection. Surviving clones were screened twice by polymerase chain reaction (PCR) and finally confirmed by Southern blot analysis before comparison. Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were confirmed by real time PCR analysis. Calcium handling potentials of these cell lines were also compared using various agonists.

RESULTS: We found significant differences in transfection efficiencies of the two cell lines (91% ± 6.1% vs 75% ± 4.2%, P = 0.01). Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for comparison. We did not observe significant differences in the levels of many known pluripotency markers. However, our genome-wide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts (P < 0.05) implicated in various important biological processes, including binding molecular functions (particularly Ca2+ binding roles). Subsequently, we measured Ca2+ signals in these cell lines in response to various calcium agonists, both in high and low Ca2+ solutions, and found significant differences (P < 0.05) in the regulation of Ca2+ homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca2+ receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines.

CONCLUSION: Results of this study emphasize the importance of considering intrinsic cellular variations, during selection of cell lines for experiments and interpretations of experimental results.

Keywords: Embryonic stem cells, Microarray, Calcium, Agonists, Transfection, Cell culture, Pluripotency, Gene targeting