Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines

To compare gene targeting efficiencies, expression profiles, and Ca(2+) handling potentials in two widely used mouse embryonic stem cell lines.

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.

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 Ca(2+) binding roles). Subsequently, we measured Ca(2+) signals in these cell lines in response to various calcium agonists, both in high and low Ca(2+) solutions, and found significant differences (P < 0.05) in the regulation of Ca(2+) homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca(2+) receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines.

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