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Protecting a transgene expression from the HAC-based vector by different chromatin insulators

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an adva...

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Autores principales: Lee, Nicholas CO, Kononenko, Artem V., Lee, Hee-Sheung, Tolkunova, Elena N., Liskovykh, Mikhail A., Masumoto, Hiroshi, Earnshaw, William C., Tomilin, Alexey N., Larionov, Vladimir, Kouprina, Natalay
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Basel 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771377/
https://www.ncbi.nlm.nih.gov/pubmed/23677492
http://dx.doi.org/10.1007/s00018-013-1362-9
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author Lee, Nicholas CO
Kononenko, Artem V.
Lee, Hee-Sheung
Tolkunova, Elena N.
Liskovykh, Mikhail A.
Masumoto, Hiroshi
Earnshaw, William C.
Tomilin, Alexey N.
Larionov, Vladimir
Kouprina, Natalay
author_facet Lee, Nicholas CO
Kononenko, Artem V.
Lee, Hee-Sheung
Tolkunova, Elena N.
Liskovykh, Mikhail A.
Masumoto, Hiroshi
Earnshaw, William C.
Tomilin, Alexey N.
Larionov, Vladimir
Kouprina, Natalay
author_sort Lee, Nicholas CO
collection PubMed
description Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-013-1362-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-37713772013-09-13 Protecting a transgene expression from the HAC-based vector by different chromatin insulators Lee, Nicholas CO Kononenko, Artem V. Lee, Hee-Sheung Tolkunova, Elena N. Liskovykh, Mikhail A. Masumoto, Hiroshi Earnshaw, William C. Tomilin, Alexey N. Larionov, Vladimir Kouprina, Natalay Cell Mol Life Sci Research Article Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-013-1362-9) contains supplementary material, which is available to authorized users. Springer Basel 2013-05-16 2013 /pmc/articles/PMC3771377/ /pubmed/23677492 http://dx.doi.org/10.1007/s00018-013-1362-9 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
spellingShingle Research Article
Lee, Nicholas CO
Kononenko, Artem V.
Lee, Hee-Sheung
Tolkunova, Elena N.
Liskovykh, Mikhail A.
Masumoto, Hiroshi
Earnshaw, William C.
Tomilin, Alexey N.
Larionov, Vladimir
Kouprina, Natalay
Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title_full Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title_fullStr Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title_full_unstemmed Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title_short Protecting a transgene expression from the HAC-based vector by different chromatin insulators
title_sort protecting a transgene expression from the hac-based vector by different chromatin insulators
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771377/
https://www.ncbi.nlm.nih.gov/pubmed/23677492
http://dx.doi.org/10.1007/s00018-013-1362-9
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