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.
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.
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Yongquan
Y
Luo
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Chengyu
C
Liu
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Trevor
T
Cerbini
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Hong
H
San
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Yongshun
Y
Lin
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Guokai
G
Chen
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Mahendra S
MS
Rao
NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA zouj2@mail.nih.gov.
Jizhong
J
Zou
D000199:Actins / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D058948:Cell Tracking; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000637:transplantation*; D051379:Mice; D009203:Myocardial Infarction / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D009245:NADH Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*
D000199:Actins; D020648:Peptide Elongation Factor 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009245:NADH Dehydrogenase; D003851:Deoxyribonucleases
AAVS1; Differentiation; Genome editing; Human induced pluripotent stem cells; Transcription activator-like effector nuclease (TALEN); Transplantation