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Article: An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells
Title | An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells |
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Authors | |
Issue Date | 2008 |
Publisher | Mary 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? |
Abstract | Self-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 Identifier | http://hdl.handle.net/10722/124933 |
ISSN | 2023 Impact Factor: 2.5 2023 SCImago Journal Rankings: 0.803 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Fu, JD | en_HK |
dc.contributor.author | Jung, Y | en_HK |
dc.contributor.author | Chan, CW | en_HK |
dc.contributor.author | Li, RA | en_HK |
dc.date.accessioned | 2010-10-31T11:02:15Z | - |
dc.date.available | 2010-10-31T11:02:15Z | - |
dc.date.issued | 2008 | en_HK |
dc.identifier.citation | Stem Cells And Development, 2008, v. 17 n. 2, p. 315-324 | en_HK |
dc.identifier.issn | 1547-3287 | en_HK |
dc.identifier.uri | http://hdl.handle.net/10722/124933 | - |
dc.description.abstract | Self-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.language | eng | en_HK |
dc.publisher | Mary Ann Liebert, Inc Publishers. The Journal's web site is located at http://www.liebertpub.com/jht | en_HK |
dc.relation.ispartof | Stem Cells and Development | en_HK |
dc.rights | This 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.mesh | Cell Differentiation - genetics | - |
dc.subject.mesh | Electrophysiology | - |
dc.subject.mesh | Embryonic Stem Cells - metabolism - physiology | - |
dc.subject.mesh | Gene Expression Regulation - drug effects | - |
dc.subject.mesh | Transgenes | - |
dc.title | An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells | en_HK |
dc.type | Article | en_HK |
dc.identifier.openurl | http://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.email | Chan, CW:camchan@hku.hk | en_HK |
dc.identifier.email | Li, RA:ronaldli@hkucc.hku.hk | en_HK |
dc.identifier.authority | Chan, CW=rp01311 | en_HK |
dc.identifier.authority | Li, RA=rp01352 | en_HK |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.1089/scd.2007.0114 | en_HK |
dc.identifier.pmid | 18447646 | en_HK |
dc.identifier.scopus | eid_2-s2.0-43049104673 | en_HK |
dc.identifier.hkuros | 183042 | en_HK |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-43049104673&selection=ref&src=s&origin=recordpage | en_HK |
dc.identifier.volume | 17 | en_HK |
dc.identifier.issue | 2 | en_HK |
dc.identifier.spage | 315 | en_HK |
dc.identifier.epage | 324 | en_HK |
dc.identifier.isi | WOS:000255597100010 | - |
dc.publisher.place | United States | en_HK |
dc.identifier.scopusauthorid | Fu, JD=7401722481 | en_HK |
dc.identifier.scopusauthorid | Jung, Y=50161845900 | en_HK |
dc.identifier.scopusauthorid | Chan, CW=12240386600 | en_HK |
dc.identifier.scopusauthorid | Li, RA=7404724466 | en_HK |
dc.identifier.issnl | 1547-3287 | - |