Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter
Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter c...
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| Veröffentlicht in: | PloS one 2008-07, Vol.3 (7), p.e2532-e2532 |
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| creator | Hsiao, Edward C Yoshinaga, Yuko Nguyen, Trieu D Musone, Stacy L Kim, Judy E Swinton, Paul Espineda, Isidro Manalac, Carlota deJong, Pieter J Conklin, Bruce R |
| description | Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library. |
| doi_str_mv | 10.1371/journal.pone.0002532 |
| format | Article |
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Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0002532</identifier><identifier>PMID: 18596956</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adenosine ; Animals ; Artificial chromosomes ; Bacteria ; Bacterial artificial chromosomes ; Beryl ; Binding sites ; Biochemistry/Experimental Biophysical Methods ; Cardiomyocytes ; Cardiovascular disease ; Cardiovascular Disorders/Congenital Heart Disease ; Cell Biology/Gene Expression ; Chemical compounds ; Chromosomes, Artificial, Bacterial ; Cloning ; Embryo cells ; Embryonic stem cells ; Embryonic Stem Cells - cytology ; Embryonic Stem Cells - metabolism ; Fluorescence ; Fluorophores ; Gene expression ; Gene sequencing ; Genes ; Genes, Reporter ; Genetic Markers ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Heart diseases ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins - genetics ; Homeodomain Proteins - metabolism ; Humans ; Journalists ; Libraries ; Melatonin ; Mice ; Mice, Transgenic ; Musculoskeletal system ; Nkx2.5 protein ; Peptides ; Photoreceptors ; Plasmids ; Polypeptides ; Protection and preservation ; Proteins ; Recombination ; Regulatory sequences ; Smooth muscle ; Stem cell transplantation ; Stem cells ; Transcription Factors - genetics ; Transcription Factors - metabolism ; Xenopus</subject><ispartof>PloS one, 2008-07, Vol.3 (7), p.e2532-e2532</ispartof><rights>COPYRIGHT 2008 Public Library of Science</rights><rights>2008 Hsiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Hsiao et al. 2008</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c693t-f62ae2972a6848a909161b4fcaeeb6d6cdc2881529da9eb0508a5d42a7a65d4a3</citedby><cites>FETCH-LOGICAL-c693t-f62ae2972a6848a909161b4fcaeeb6d6cdc2881529da9eb0508a5d42a7a65d4a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430532/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430532/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23864,27922,27923,53789,53791,79370,79371</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18596956$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Najbauer, Joseph</contributor><creatorcontrib>Hsiao, Edward C</creatorcontrib><creatorcontrib>Yoshinaga, Yuko</creatorcontrib><creatorcontrib>Nguyen, Trieu D</creatorcontrib><creatorcontrib>Musone, Stacy L</creatorcontrib><creatorcontrib>Kim, Judy E</creatorcontrib><creatorcontrib>Swinton, Paul</creatorcontrib><creatorcontrib>Espineda, Isidro</creatorcontrib><creatorcontrib>Manalac, Carlota</creatorcontrib><creatorcontrib>deJong, Pieter J</creatorcontrib><creatorcontrib>Conklin, Bruce R</creatorcontrib><title>Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
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The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.</description><subject>Adenosine</subject><subject>Animals</subject><subject>Artificial chromosomes</subject><subject>Bacteria</subject><subject>Bacterial artificial chromosomes</subject><subject>Beryl</subject><subject>Binding sites</subject><subject>Biochemistry/Experimental Biophysical Methods</subject><subject>Cardiomyocytes</subject><subject>Cardiovascular disease</subject><subject>Cardiovascular Disorders/Congenital Heart Disease</subject><subject>Cell Biology/Gene Expression</subject><subject>Chemical compounds</subject><subject>Chromosomes, Artificial, Bacterial</subject><subject>Cloning</subject><subject>Embryo cells</subject><subject>Embryonic stem cells</subject><subject>Embryonic Stem Cells - cytology</subject><subject>Embryonic Stem Cells - 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embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter</title><author>Hsiao, Edward C ; Yoshinaga, Yuko ; Nguyen, Trieu D ; Musone, Stacy L ; Kim, Judy E ; Swinton, Paul ; Espineda, Isidro ; Manalac, Carlota ; deJong, Pieter J ; Conklin, Bruce R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c693t-f62ae2972a6848a909161b4fcaeeb6d6cdc2881529da9eb0508a5d42a7a65d4a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Adenosine</topic><topic>Animals</topic><topic>Artificial chromosomes</topic><topic>Bacteria</topic><topic>Bacterial artificial chromosomes</topic><topic>Beryl</topic><topic>Binding sites</topic><topic>Biochemistry/Experimental Biophysical Methods</topic><topic>Cardiomyocytes</topic><topic>Cardiovascular disease</topic><topic>Cardiovascular Disorders/Congenital Heart Disease</topic><topic>Cell Biology/Gene 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Carlota</au><au>deJong, Pieter J</au><au>Conklin, Bruce R</au><au>Najbauer, Joseph</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2008-07-02</date><risdate>2008</risdate><volume>3</volume><issue>7</issue><spage>e2532</spage><epage>e2532</epage><pages>e2532-e2532</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.
Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.
Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>18596956</pmid><doi>10.1371/journal.pone.0002532</doi><tpages>e2532</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2008-07, Vol.3 (7), p.e2532-e2532 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1312319458 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Adenosine Animals Artificial chromosomes Bacteria Bacterial artificial chromosomes Beryl Binding sites Biochemistry/Experimental Biophysical Methods Cardiomyocytes Cardiovascular disease Cardiovascular Disorders/Congenital Heart Disease Cell Biology/Gene Expression Chemical compounds Chromosomes, Artificial, Bacterial Cloning Embryo cells Embryonic stem cells Embryonic Stem Cells - cytology Embryonic Stem Cells - metabolism Fluorescence Fluorophores Gene expression Gene sequencing Genes Genes, Reporter Genetic Markers Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Heart diseases Homeobox Protein Nkx-2.5 Homeodomain Proteins - genetics Homeodomain Proteins - metabolism Humans Journalists Libraries Melatonin Mice Mice, Transgenic Musculoskeletal system Nkx2.5 protein Peptides Photoreceptors Plasmids Polypeptides Protection and preservation Proteins Recombination Regulatory sequences Smooth muscle Stem cell transplantation Stem cells Transcription Factors - genetics Transcription Factors - metabolism Xenopus |
| title | Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-09T23%3A22%3A08IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Marking%20embryonic%20stem%20cells%20with%20a%202A%20self-cleaving%20peptide:%20a%20NKX2-5%20emerald%20GFP%20BAC%20reporter&rft.jtitle=PloS%20one&rft.au=Hsiao,%20Edward%20C&rft.date=2008-07-02&rft.volume=3&rft.issue=7&rft.spage=e2532&rft.epage=e2532&rft.pages=e2532-e2532&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0002532&rft_dat=%3Cgale_plos_%3EA472641711%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1312319458&rft_id=info:pmid/18596956&rft_galeid=A472641711&rft_doaj_id=oai_doaj_org_article_ee98f97fbb37487691ed66b2f176cc32&rfr_iscdi=true |