基因在胚胎的干细胞衬里的老鼠指向和钙处理效率

• 作者: Solomon Mamo Julianna Kobolak Istvan Borbíró Tamás Bíró Istvan Bock Andras Dinnyes
• 来源期刊: 世界干细胞杂志：英文版（电子版）
• 学科: 生物学
• 关键词: EMBRYONIC stem cells MICROARRAY Calcium AGONISTS TRANSFECTION Cell culture PLURIPOTENCY Gene targeting
• 年，卷（期）: 2010,（6）
• 页码范围: 127-140
• 页数: 14页
• 分类号: Q813

摘要

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 conf irmed by real time PCR analysis.Calcium handling potentials of these cell lines were also compared using various agonists.RESULTS: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 comparison.We did not observe signif icant differences in the levels of many known pluripotency markers.However,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 between the investigated cell lines.Then we f