In mice,gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics.This strategy allows precise location of the site of transgene integration and is most...In mice,gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics.This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression("knock-out"),or to introduce mutant or modified alleles at the locus of interest("knock-in").The efficacy of producing live,transgenic mice challenges our understanding of this complex process,and of the factors which influence germline competence of embryonic stem cell lines.Increasingly,evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell(ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability.Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential.There are a number of options for generating chimeras from ES cells (ES chimera mice);however,each method has its advantages and disadvantages.Recent developments in induced pluripotent stem(iPS)cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies.The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines.The most recent methods for generating specifically ES and iPS chimera mice,including the advantages and disadvantages of each method are also discussed.展开更多
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,we...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 further compared the detection and expression of various membrane and in-tracellular Ca2+ receptors and similarly found signif icant(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.展开更多
基金Supported by Grants from EU FP6("MEDRAT"-LSHG-CT-2005-518240"Artemis",LSHM-CT-2006-037862+5 种基金"AGLAEA",LSHM-CT-2006-037554,"CLONET",MRTN-CT-2006-035468),EU FP7("PartnErS",PIAP-GA-2008-218205"InduHeart",EUFP7-PEOPL E-IRG-2008-234390"InduStem",PIAP-GA-2008-230675"Plurisys",HEALTH-F4-2009-223485)NKFP_07_1-ES2HEART-HU,No.OM-00202-2007 NKTH/ANRTET Franco-Hungarian Bilateral Scientific and Technological Collaborative Project"Plurabit"
文摘In mice,gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics.This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression("knock-out"),or to introduce mutant or modified alleles at the locus of interest("knock-in").The efficacy of producing live,transgenic mice challenges our understanding of this complex process,and of the factors which influence germline competence of embryonic stem cell lines.Increasingly,evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell(ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability.Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential.There are a number of options for generating chimeras from ES cells (ES chimera mice);however,each method has its advantages and disadvantages.Recent developments in induced pluripotent stem(iPS)cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies.The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines.The most recent methods for generating specifically ES and iPS chimera mice,including the advantages and disadvantages of each method are also discussed.
文摘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 further compared the detection and expression of various membrane and in-tracellular Ca2+ receptors and similarly found signif icant(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.
