The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal trans...

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Autores principales:	Nynne Sharma, Anne Kruse Hollensen, Rasmus O Bak, Nicklas Heine Staunstrup, Lisbeth Dahl Schrøder, Jacob Giehm Mikkelsen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ae30b14198b642d5b0ac2f89ee7ed5d8
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spelling	oai:doaj.org-article:ae30b14198b642d5b0ac2f89ee7ed5d82021-11-18T08:10:49ZThe impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.1932-620310.1371/journal.pone.0048421https://doaj.org/article/ae30b14198b642d5b0ac2f89ee7ed5d82012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23110238/?tool=EBIhttps://doaj.org/toc/1932-6203DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.Nynne SharmaAnne Kruse HollensenRasmus O BakNicklas Heine StaunstrupLisbeth Dahl SchrøderJacob Giehm MikkelsenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 10, p e48421 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nynne Sharma Anne Kruse Hollensen Rasmus O Bak Nicklas Heine Staunstrup Lisbeth Dahl Schrøder Jacob Giehm Mikkelsen The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
description	DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.
format	article
author	Nynne Sharma Anne Kruse Hollensen Rasmus O Bak Nicklas Heine Staunstrup Lisbeth Dahl Schrøder Jacob Giehm Mikkelsen
author_facet	Nynne Sharma Anne Kruse Hollensen Rasmus O Bak Nicklas Heine Staunstrup Lisbeth Dahl Schrøder Jacob Giehm Mikkelsen
author_sort	Nynne Sharma
title	The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
title_short	The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
title_full	The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
title_fullStr	The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
title_full_unstemmed	The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.
title_sort	impact of chs4 insulators on dna transposon vector mobilization and silencing in retinal pigment epithelium cells.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/ae30b14198b642d5b0ac2f89ee7ed5d8
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The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

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