Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases
The authors describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high‐efficiency targeting can be obtained with open‐source TALENs and that caref...
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description | The authors describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Human induced pluripotent stem (hiPS) cell lines with tissue‐specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno‐associated virus integration site 1 (AAVS1) locus has been used as a “safe harbor” locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus‐immediate early enhancer/chicken β‐actin/rabbit β‐globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage‐specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1‐CAG‐EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo. |
doi_str_mv | 10.5966/sctm.2013-0212 |
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fullrecord | <record><control><sourceid>proquest_24P</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4073825</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2290592709</sourcerecordid><originalsourceid>FETCH-LOGICAL-c5341-6072fa76b58d02ec94660d8e8e5372bb8be0ef36a264b707596614436a2e94de3</originalsourceid><addsrcrecordid>eNqFks9u1DAQxiMEolXplSOyxHkX_0mc5IIULdttpRVU7MLVcpIJdXHsYDste-MReDiegCfBIWUFJ3yxZ_zzN5_GkyTPCV5mJeevfBP6JcWELTAl9FFySkmWL3hW4MfHM09PknPvb3FcvOQlxU-TE5oWjGUlOU1-7IKsNaC1uZGmgRZtHIBBF3q0DnwDJqBrZwMog9Zfh5jyyhpUdQEceqO6DlxElAxTVpoW7Z00ftDShDlnO_QeBusm_nLspUFXph2nQtd6dGqI0rHELkCPVqC1Rxsw4GSIQH1AVfVxR2bNJsK_FasmqDsZrPv57ftWfY7Wo4smxujt2GiQHvyz5EkntYfzh_0s-XCx3q8uF9t3m6tVtV00GUvJguOcdjLndVa0mEJTppzjtoACMpbTui5qwNAxLilP6xznU89Jmk4JKNMW2FnyetYdxrqHduqWk1oMTvXSHYSVSvx7Y9SN-GTvRIpzVtAsCrx8EHD2ywg-iFs7OhM9C0pLnJU0x2WkljPVOOu9g-5YgWAxmRLTIIhpEMQ0CPHBi799HfE_3x6BcgbulYbDf-TEbrVnMUpZXlDCfgGvq8a2</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2290592709</pqid></control><display><type>article</type><title>Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases</title><source>Wiley Online Library (Open Access Collection)</source><creator>Luo, Yongquan ; Liu, Chengyu ; Cerbini, Trevor ; San, Hong ; Lin, Yongshun ; Chen, Guokai ; Rao, Mahendra S. ; Zou, Jizhong</creator><creatorcontrib>Luo, Yongquan ; Liu, Chengyu ; Cerbini, Trevor ; San, Hong ; Lin, Yongshun ; Chen, Guokai ; Rao, Mahendra S. ; Zou, Jizhong</creatorcontrib><description>The authors describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Human induced pluripotent stem (hiPS) cell lines with tissue‐specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno‐associated virus integration site 1 (AAVS1) locus has been used as a “safe harbor” locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus‐immediate early enhancer/chicken β‐actin/rabbit β‐globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage‐specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1‐CAG‐EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.</description><identifier>ISSN: 2157-6564</identifier><identifier>EISSN: 2157-6580</identifier><identifier>DOI: 10.5966/sctm.2013-0212</identifier><identifier>PMID: 24833591</identifier><language>eng</language><publisher>Durham, NC, USA: AlphaMed Press</publisher><subject>AAVS1 ; Actins - genetics ; Amino acids ; Animals ; Cardiomyocytes ; Cell Differentiation ; Cell Lineage ; Cell Tracking ; Cells, Cultured ; Cytomegalovirus - genetics ; Data analysis ; Deoxyribonucleases - metabolism ; Deoxyribonucleic acid ; Dependovirus - genetics ; Design ; Differentiation ; Disease Models, Animal ; DNA ; Enabling Technologies for Cell-Based Clinical Translation ; Gene expression ; Gene Expression Regulation ; Gene Silencing ; Genes, Reporter ; Genome editing ; Genomes ; Green fluorescent protein ; Green Fluorescent Proteins - biosynthesis ; Green Fluorescent Proteins - genetics ; Heart ; Human induced pluripotent stem cells ; Humans ; Induced Pluripotent Stem Cells - metabolism ; Induced Pluripotent Stem Cells - transplantation ; Mice ; Myocardial Infarction - genetics ; Myocardial Infarction - metabolism ; Myocardial Infarction - pathology ; Myocardial Infarction - surgery ; Myocardium - metabolism ; Myocardium - pathology ; Myocytes, Cardiac - metabolism ; Myocytes, Cardiac - pathology ; Myocytes, Cardiac - transplantation ; NADH Dehydrogenase - biosynthesis ; NADH Dehydrogenase - genetics ; Nonprofit organizations ; Nuclease ; Peptide Elongation Factor 1 - genetics ; Pluripotency ; Promoter Regions, Genetic ; Proteins ; Stem cells ; Studies ; Time Factors ; Transcription ; Transcription activator-like effector nuclease (TALEN) ; Transcription activator-like effector nucleases ; Transduction, Genetic ; Transfection - methods ; Transplantation ; Writing ; Zinc finger proteins</subject><ispartof>Stem cells translational medicine, 2014-07, Vol.3 (7), p.821-835</ispartof><rights>2014 AlphaMed Press</rights><rights>AlphaMed Press.</rights><rights>2014. This work is published under https://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>AlphaMed Press 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5341-6072fa76b58d02ec94660d8e8e5372bb8be0ef36a264b707596614436a2e94de3</citedby><cites>FETCH-LOGICAL-c5341-6072fa76b58d02ec94660d8e8e5372bb8be0ef36a264b707596614436a2e94de3</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/PMC4073825/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073825/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,315,728,781,785,865,886,1418,11567,27929,27930,45579,45580,46057,46481,53796,53798</link.rule.ids><linktorsrc>$$Uhttps://onlinelibrary.wiley.com/doi/abs/10.5966%2Fsctm.2013-0212$$EView_record_in_Wiley-Blackwell$$FView_record_in_$$GWiley-Blackwell</linktorsrc><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24833591$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Luo, Yongquan</creatorcontrib><creatorcontrib>Liu, Chengyu</creatorcontrib><creatorcontrib>Cerbini, Trevor</creatorcontrib><creatorcontrib>San, Hong</creatorcontrib><creatorcontrib>Lin, Yongshun</creatorcontrib><creatorcontrib>Chen, Guokai</creatorcontrib><creatorcontrib>Rao, Mahendra S.</creatorcontrib><creatorcontrib>Zou, Jizhong</creatorcontrib><title>Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases</title><title>Stem cells translational medicine</title><addtitle>Stem Cells Transl Med</addtitle><description>The authors describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Human induced pluripotent stem (hiPS) cell lines with tissue‐specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno‐associated virus integration site 1 (AAVS1) locus has been used as a “safe harbor” locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus‐immediate early enhancer/chicken β‐actin/rabbit β‐globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage‐specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1‐CAG‐EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.</description><subject>AAVS1</subject><subject>Actins - genetics</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Cardiomyocytes</subject><subject>Cell Differentiation</subject><subject>Cell Lineage</subject><subject>Cell Tracking</subject><subject>Cells, Cultured</subject><subject>Cytomegalovirus - genetics</subject><subject>Data analysis</subject><subject>Deoxyribonucleases - metabolism</subject><subject>Deoxyribonucleic acid</subject><subject>Dependovirus - genetics</subject><subject>Design</subject><subject>Differentiation</subject><subject>Disease Models, Animal</subject><subject>DNA</subject><subject>Enabling Technologies for Cell-Based Clinical Translation</subject><subject>Gene expression</subject><subject>Gene Expression Regulation</subject><subject>Gene Silencing</subject><subject>Genes, Reporter</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>Green fluorescent protein</subject><subject>Green Fluorescent Proteins - biosynthesis</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Heart</subject><subject>Human induced pluripotent stem cells</subject><subject>Humans</subject><subject>Induced Pluripotent Stem Cells - metabolism</subject><subject>Induced Pluripotent Stem Cells - transplantation</subject><subject>Mice</subject><subject>Myocardial Infarction - genetics</subject><subject>Myocardial Infarction - metabolism</subject><subject>Myocardial Infarction - pathology</subject><subject>Myocardial Infarction - surgery</subject><subject>Myocardium - metabolism</subject><subject>Myocardium - pathology</subject><subject>Myocytes, Cardiac - metabolism</subject><subject>Myocytes, Cardiac - pathology</subject><subject>Myocytes, Cardiac - transplantation</subject><subject>NADH Dehydrogenase - biosynthesis</subject><subject>NADH Dehydrogenase - genetics</subject><subject>Nonprofit organizations</subject><subject>Nuclease</subject><subject>Peptide Elongation Factor 1 - genetics</subject><subject>Pluripotency</subject><subject>Promoter Regions, Genetic</subject><subject>Proteins</subject><subject>Stem cells</subject><subject>Studies</subject><subject>Time Factors</subject><subject>Transcription</subject><subject>Transcription activator-like effector nuclease (TALEN)</subject><subject>Transcription activator-like effector nucleases</subject><subject>Transduction, Genetic</subject><subject>Transfection - methods</subject><subject>Transplantation</subject><subject>Writing</subject><subject>Zinc finger proteins</subject><issn>2157-6564</issn><issn>2157-6580</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqFks9u1DAQxiMEolXplSOyxHkX_0mc5IIULdttpRVU7MLVcpIJdXHsYDste-MReDiegCfBIWUFJ3yxZ_zzN5_GkyTPCV5mJeevfBP6JcWELTAl9FFySkmWL3hW4MfHM09PknPvb3FcvOQlxU-TE5oWjGUlOU1-7IKsNaC1uZGmgRZtHIBBF3q0DnwDJqBrZwMog9Zfh5jyyhpUdQEceqO6DlxElAxTVpoW7Z00ftDShDlnO_QeBusm_nLspUFXph2nQtd6dGqI0rHELkCPVqC1Rxsw4GSIQH1AVfVxR2bNJsK_FasmqDsZrPv57ftWfY7Wo4smxujt2GiQHvyz5EkntYfzh_0s-XCx3q8uF9t3m6tVtV00GUvJguOcdjLndVa0mEJTppzjtoACMpbTui5qwNAxLilP6xznU89Jmk4JKNMW2FnyetYdxrqHduqWk1oMTvXSHYSVSvx7Y9SN-GTvRIpzVtAsCrx8EHD2ywg-iFs7OhM9C0pLnJU0x2WkljPVOOu9g-5YgWAxmRLTIIhpEMQ0CPHBi799HfE_3x6BcgbulYbDf-TEbrVnMUpZXlDCfgGvq8a2</recordid><startdate>201407</startdate><enddate>201407</enddate><creator>Luo, Yongquan</creator><creator>Liu, Chengyu</creator><creator>Cerbini, Trevor</creator><creator>San, Hong</creator><creator>Lin, Yongshun</creator><creator>Chen, Guokai</creator><creator>Rao, Mahendra S.</creator><creator>Zou, Jizhong</creator><general>AlphaMed Press</general><general>Oxford University Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>5PM</scope></search><sort><creationdate>201407</creationdate><title>Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases</title><author>Luo, Yongquan ; Liu, Chengyu ; Cerbini, Trevor ; San, Hong ; Lin, Yongshun ; Chen, Guokai ; Rao, Mahendra S. ; Zou, Jizhong</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5341-6072fa76b58d02ec94660d8e8e5372bb8be0ef36a264b707596614436a2e94de3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>AAVS1</topic><topic>Actins - genetics</topic><topic>Amino acids</topic><topic>Animals</topic><topic>Cardiomyocytes</topic><topic>Cell Differentiation</topic><topic>Cell Lineage</topic><topic>Cell Tracking</topic><topic>Cells, Cultured</topic><topic>Cytomegalovirus - genetics</topic><topic>Data analysis</topic><topic>Deoxyribonucleases - metabolism</topic><topic>Deoxyribonucleic acid</topic><topic>Dependovirus - genetics</topic><topic>Design</topic><topic>Differentiation</topic><topic>Disease Models, Animal</topic><topic>DNA</topic><topic>Enabling Technologies for Cell-Based Clinical Translation</topic><topic>Gene expression</topic><topic>Gene Expression Regulation</topic><topic>Gene Silencing</topic><topic>Genes, Reporter</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>Green fluorescent protein</topic><topic>Green Fluorescent Proteins - biosynthesis</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Heart</topic><topic>Human induced pluripotent stem cells</topic><topic>Humans</topic><topic>Induced Pluripotent Stem Cells - 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methods</topic><topic>Transplantation</topic><topic>Writing</topic><topic>Zinc finger proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Luo, Yongquan</creatorcontrib><creatorcontrib>Liu, Chengyu</creatorcontrib><creatorcontrib>Cerbini, Trevor</creatorcontrib><creatorcontrib>San, Hong</creatorcontrib><creatorcontrib>Lin, Yongshun</creatorcontrib><creatorcontrib>Chen, Guokai</creatorcontrib><creatorcontrib>Rao, Mahendra S.</creatorcontrib><creatorcontrib>Zou, Jizhong</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Biological Science Database</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Stem cells translational medicine</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Luo, Yongquan</au><au>Liu, Chengyu</au><au>Cerbini, Trevor</au><au>San, Hong</au><au>Lin, Yongshun</au><au>Chen, Guokai</au><au>Rao, Mahendra S.</au><au>Zou, Jizhong</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases</atitle><jtitle>Stem cells translational medicine</jtitle><addtitle>Stem Cells Transl Med</addtitle><date>2014-07</date><risdate>2014</risdate><volume>3</volume><issue>7</issue><spage>821</spage><epage>835</epage><pages>821-835</pages><issn>2157-6564</issn><eissn>2157-6580</eissn><abstract>The authors describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.
Human induced pluripotent stem (hiPS) cell lines with tissue‐specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno‐associated virus integration site 1 (AAVS1) locus has been used as a “safe harbor” locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator‐like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus‐immediate early enhancer/chicken β‐actin/rabbit β‐globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage‐specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1‐CAG‐EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high‐efficiency targeting can be obtained with open‐source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.</abstract><cop>Durham, NC, USA</cop><pub>AlphaMed Press</pub><pmid>24833591</pmid><doi>10.5966/sctm.2013-0212</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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subjects | AAVS1 Actins - genetics Amino acids Animals Cardiomyocytes Cell Differentiation Cell Lineage Cell Tracking Cells, Cultured Cytomegalovirus - genetics Data analysis Deoxyribonucleases - metabolism Deoxyribonucleic acid Dependovirus - genetics Design Differentiation Disease Models, Animal DNA Enabling Technologies for Cell-Based Clinical Translation Gene expression Gene Expression Regulation Gene Silencing Genes, Reporter Genome editing Genomes Green fluorescent protein Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - genetics Heart Human induced pluripotent stem cells Humans Induced Pluripotent Stem Cells - metabolism Induced Pluripotent Stem Cells - transplantation Mice Myocardial Infarction - genetics Myocardial Infarction - metabolism Myocardial Infarction - pathology Myocardial Infarction - surgery Myocardium - metabolism Myocardium - pathology Myocytes, Cardiac - metabolism Myocytes, Cardiac - pathology Myocytes, Cardiac - transplantation NADH Dehydrogenase - biosynthesis NADH Dehydrogenase - genetics Nonprofit organizations Nuclease Peptide Elongation Factor 1 - genetics Pluripotency Promoter Regions, Genetic Proteins Stem cells Studies Time Factors Transcription Transcription activator-like effector nuclease (TALEN) Transcription activator-like effector nucleases Transduction, Genetic Transfection - methods Transplantation Writing Zinc finger proteins |
title | Stable Enhanced Green Fluorescent Protein Expression After Differentiation and Transplantation of Reporter Human Induced Pluripotent Stem Cells Generated by AAVS1 Transcription Activator‐Like Effector Nucleases |
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