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Article: An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells

TitleAn inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells
Authors
Issue Date2008
PublisherMary Ann Liebert, Inc Publishers. The Journal's web site is located at http://www.liebertpub.com/jht
Citation
Stem Cells And Development, 2008, v. 17 n. 2, p. 315-324 How to Cite?
AbstractSelf-renewing pluripotent human embryonic stem (hES) cells are capable of regenerating such non-dividing cells as neurons and cardiomyocytes for therapies and can serve as an excellent experimental model for studying early human development. Both the spatial and temporal relationships of gene expression play a crucial role in determining differentiation; to obtain a better understanding of hES cell differentiation, it will be necessary to establish an inducible system in hES cells that enables specific transgene(s) to reversibly and conditionally express (1) at specific levels and (2) at particular time points during development. Using lentivirus (LV)-mediated gene transfer and a tetracycline-controlled trans-repressor (TR), we first established in hES cells a doxycycline (DOX)-inducible expression system of green fluorescent protein (GFP) to probe its reversibility and kinetics. Upon the addition of DOX, the percentage of GFP + hES cells increased time dependently: The time at which 50% of all green cells appeared (T 50 on) was 119.5 ± 3.2 h; upon DOX removal, GFP expression declined with a half-time (T 50 off) of 127.7 ± 3.9 h and became completely silenced at day 8. Both the proportion and total mean fluorescence intensity (MFI) were dose-dependent (EC 50 = 24.5 ± 2.2 ng/ml). The same system when incorporated into murine (m) ES cells similarly exhibited reversible dose-dependent responses with a similar sensitivity (EC 50 =49.5 ± 8.5 ng/ml), but the much faster kinetics (T 50 on = 35.5 ± 5.5 h, T 50 off = 71.5 ± 2.4 hours). DOX-induced expression of the Kir2.1 channels in mES and hES cells led to robust expression of the inwardly rectifying potassium (K +) current and thereby hyperpolarized the resting membrane potential (RMP). We conclude that the LV-inducible system established presents a unique tool for probing differentiation. © 2008 Mary Ann Liebert, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/124933
ISSN
2015 Impact Factor: 3.777
2015 SCImago Journal Rankings: 1.703
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorFu, JDen_HK
dc.contributor.authorJung, Yen_HK
dc.contributor.authorChan, CWen_HK
dc.contributor.authorLi, RAen_HK
dc.date.accessioned2010-10-31T11:02:15Z-
dc.date.available2010-10-31T11:02:15Z-
dc.date.issued2008en_HK
dc.identifier.citationStem Cells And Development, 2008, v. 17 n. 2, p. 315-324en_HK
dc.identifier.issn1547-3287en_HK
dc.identifier.urihttp://hdl.handle.net/10722/124933-
dc.description.abstractSelf-renewing pluripotent human embryonic stem (hES) cells are capable of regenerating such non-dividing cells as neurons and cardiomyocytes for therapies and can serve as an excellent experimental model for studying early human development. Both the spatial and temporal relationships of gene expression play a crucial role in determining differentiation; to obtain a better understanding of hES cell differentiation, it will be necessary to establish an inducible system in hES cells that enables specific transgene(s) to reversibly and conditionally express (1) at specific levels and (2) at particular time points during development. Using lentivirus (LV)-mediated gene transfer and a tetracycline-controlled trans-repressor (TR), we first established in hES cells a doxycycline (DOX)-inducible expression system of green fluorescent protein (GFP) to probe its reversibility and kinetics. Upon the addition of DOX, the percentage of GFP + hES cells increased time dependently: The time at which 50% of all green cells appeared (T 50 on) was 119.5 ± 3.2 h; upon DOX removal, GFP expression declined with a half-time (T 50 off) of 127.7 ± 3.9 h and became completely silenced at day 8. Both the proportion and total mean fluorescence intensity (MFI) were dose-dependent (EC 50 = 24.5 ± 2.2 ng/ml). The same system when incorporated into murine (m) ES cells similarly exhibited reversible dose-dependent responses with a similar sensitivity (EC 50 =49.5 ± 8.5 ng/ml), but the much faster kinetics (T 50 on = 35.5 ± 5.5 h, T 50 off = 71.5 ± 2.4 hours). DOX-induced expression of the Kir2.1 channels in mES and hES cells led to robust expression of the inwardly rectifying potassium (K +) current and thereby hyperpolarized the resting membrane potential (RMP). We conclude that the LV-inducible system established presents a unique tool for probing differentiation. © 2008 Mary Ann Liebert, Inc.en_HK
dc.languageengen_HK
dc.publisherMary Ann Liebert, Inc Publishers. The Journal's web site is located at http://www.liebertpub.com/jhten_HK
dc.relation.ispartofStem Cells and Developmenten_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThis is a copy of an article published in the Stem Cells and Development © 2008 Copyright Mary Ann Liebert, Inc Publishers ; Stem Cells and Development is available online at: http://www.liebertonline.com-
dc.subject.meshCell Differentiation - genetics-
dc.subject.meshElectrophysiology-
dc.subject.meshEmbryonic Stem Cells - metabolism - physiology-
dc.subject.meshGene Expression Regulation - drug effects-
dc.subject.meshTransgenes-
dc.titleAn inducible transgene expression system for regulated phenotypic modification of human embryonic stem cellsen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1547-3287&volume=17&issue=2&spage=315&epage=324&date=2008&atitle=An+inducible+transgene+expression+system+for+regulated+phenotypic+modification+of+human+embryonic+stem+cells-
dc.identifier.emailChan, CW:camchan@hku.hken_HK
dc.identifier.emailLi, RA:ronaldli@hkucc.hku.hken_HK
dc.identifier.authorityChan, CW=rp01311en_HK
dc.identifier.authorityLi, RA=rp01352en_HK
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1089/scd.2007.0114en_HK
dc.identifier.pmid18447646en_HK
dc.identifier.scopuseid_2-s2.0-43049104673en_HK
dc.identifier.hkuros183042en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-43049104673&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume17en_HK
dc.identifier.issue2en_HK
dc.identifier.spage315en_HK
dc.identifier.epage324en_HK
dc.identifier.isiWOS:000255597100010-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridFu, JD=7401722481en_HK
dc.identifier.scopusauthoridJung, Y=50161845900en_HK
dc.identifier.scopusauthoridChan, CW=12240386600en_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK

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