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

Overview

Journal Stem Cells Transl Med

Publisher Oxford University Press

Specialties Cell Biology
Pharmacology

Date 2014 May 17

PMID 24833591

Citations 50

Authors

Yongquan Luo

Chengyu Liu

Trevor Cerbini

Hong San

Yongshun Lin

Guokai Chen

Mahendra S Rao

Jizhong Zou

Affiliations

Soon will be listed here.

Abstract

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.

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References

Rao M, Malik N . Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012; 113(10):3061-8. PMC: 3552293. DOI: 10.1002/jcb.24183. View

Chou B, Mali P, Huang X, Ye Z, Dowey S, Resar L . Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res. 2011; 21(3):518-29. PMC: 3193421. DOI: 10.1038/cr.2011.12. View

Xia X, Zhang Y, Zieth C, Zhang S . Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner. Stem Cells Dev. 2007; 16(1):167-76. PMC: 2801347. DOI: 10.1089/scd.2006.0057. View

cermak T, Doyle E, Christian M, Wang L, Zhang Y, Schmidt C . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39(12):e82. PMC: 3130291. DOI: 10.1093/nar/gkr218. View

Zou J, Maeder M, Mali P, Pruett-Miller S, Thibodeau-Beganny S, Chou B . Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell. 2009; 5(1):97-110. PMC: 2720132. DOI: 10.1016/j.stem.2009.05.023. View

Sanjana N, Cong L, Zhou Y, Cunniff M, Feng G, Zhang F . A transcription activator-like effector toolbox for genome engineering. Nat Protoc. 2012; 7(1):171-92. PMC: 3684555. DOI: 10.1038/nprot.2011.431. View

Mayer-Proschel M, Kalyani A, Mujtaba T, Rao M . Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron. 1997; 19(4):773-85. DOI: 10.1016/s0896-6273(00)80960-5. View

Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver R . Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol. 2009; 27(9):851-7. PMC: 4142824. DOI: 10.1038/nbt.1562. View

Chen G, Gulbranson D, Hou Z, Bolin J, Ruotti V, Probasco M . Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011; 8(5):424-9. PMC: 3084903. DOI: 10.1038/nmeth.1593. View

10.

Boch J, Bonas U . Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol. 2009; 48:419-36. DOI: 10.1146/annurev-phyto-080508-081936. View

11.

DeKelver R, Choi V, Moehle E, Paschon D, Hockemeyer D, Meijsing S . Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 2010; 20(8):1133-42. PMC: 2909576. DOI: 10.1101/gr.106773.110. View

12.

MacArthur C, Xue H, Van Hoof D, Lieu P, Dudas M, Fontes A . Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus. Stem Cells Dev. 2011; 21(2):191-205. PMC: 3258440. DOI: 10.1089/scd.2011.0163. View

13.

Walia B, Satija N, Tripathi R, Gangenahalli G . Induced pluripotent stem cells: fundamentals and applications of the reprogramming process and its ramifications on regenerative medicine. Stem Cell Rev Rep. 2011; 8(1):100-15. DOI: 10.1007/s12015-011-9279-x. View

14.

Zou J, Sweeney C, Chou B, Choi U, Pan J, Wang H . Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood. 2011; 117(21):5561-72. PMC: 3110021. DOI: 10.1182/blood-2010-12-328161. View

15.

Lamartina S, Sporeno E, Fattori E, Toniatti C . Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment. J Virol. 2000; 74(16):7671-7. PMC: 112291. DOI: 10.1128/jvi.74.16.7671-7677.2000. View

16.

Smith J, Maguire S, Davis L, Alexander M, Yang F, Chandran S . Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells. 2007; 26(2):496-504. DOI: 10.1634/stemcells.2007-0039. View

17.

Ogata T, Kozuka T, Kanda T . Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J Virol. 2003; 77(16):9000-7. PMC: 167236. DOI: 10.1128/jvi.77.16.9000-9007.2003. View

18.

Miller J, Tan S, Qiao G, Barlow K, Wang J, Xia D . A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2010; 29(2):143-8. DOI: 10.1038/nbt.1755. View

19.

Ng S, Wong C, Tsang S . Differential gene expressions in atrial and ventricular myocytes: insights into the road of applying embryonic stem cell-derived cardiomyocytes for future therapies. Am J Physiol Cell Physiol. 2010; 299(6):C1234-49. DOI: 10.1152/ajpcell.00402.2009. View

20.

Bronson S, Plaehn E, Kluckman K, Hagaman J, Maeda N, SMITHIES O . Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci U S A. 1996; 93(17):9067-72. PMC: 38596. DOI: 10.1073/pnas.93.17.9067. View