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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Veröffentlicht in:Stem cells translational medicine 2014-07, Vol.3 (7), p.821-835
Hauptverfasser: Luo, Yongquan, Liu, Chengyu, Cerbini, Trevor, San, Hong, Lin, Yongshun, Chen, Guokai, Rao, Mahendra S., Zou, Jizhong
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container_end_page 835
container_issue 7
container_start_page 821
container_title Stem cells translational medicine
container_volume 3
creator Luo, Yongquan
Liu, Chengyu
Cerbini, Trevor
San, Hong
Lin, Yongshun
Chen, Guokai
Rao, Mahendra S.
Zou, Jizhong
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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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. 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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. 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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 - 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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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source Wiley Online Library (Open Access Collection)
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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