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

World Journal of Stem Cells 2010年第6期2卷 127-140页

作者： Solomon Mamo julianna kobolak Istvan Borbíró Tamás Bíró Istvan Bock Andras Dinnyes Genetic Reprogramming Group Agricultural Biotechnology CenterSzent-Gyorgyi Albert ut.4H-2100 GdllHungary Solomon MamoLyons Research FarmUniversity College DublinNewcastleCo DublinIreland Department of Physiology Research Center for Molecular MedicineMedical and Health Science CenterUniversity of DebrecenNagyerdei krt.98H-4032 DebrecenHungary Abiol Ltd. Nagyerdei krt.98H-4032 DebrecenHungary BioTalentum Ltd. Aulich l.26GdllHungary Andras DinnyesMolecular Animal Biotechnology LaboratorySzent Istvan UniversityPáter K.u.1H-2103 GdllHungary

AIM:To compare gene targeting efficiencies,expression profiles,and Ca2+ handling potentials in two widely used mouse embryonic stem cell ***: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 *** were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency ***,cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct,following the BioRad and Amaxa transfection ***,the cells were cultured and neomycin-resistant cells were picked after 13 d of *** clones were screened twice by polymerase chain reaction(PCR) and finally confirmed by Southern blot analysis before *** gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were conf irmed by real time PCR *** handling potentials of these cell lines were also compared using various ***:We found signif icant differences in transfection eff iciencies 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 *** did not observe signif icant differences in the levels of many known pluripotency ***,our genomewide 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 signif icant differences(P < 0.05) in the regulation of Ca2+ homeostasis betwee

关键词： Embryonic stem cells Microarray Calcium Agonists Transfection Cell culture Pluripotency Gene targeting

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