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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Bibliographic Details
Published in:Cellular and molecular life sciences : CMLS 2013-10, Vol.70 (19), p.3723-3737
Main Authors: 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
Format: Article
Language:English
Subjects:
Animals
Biochemistry
Biomedical and Life Sciences
Biomedicine
Cell Biology
Cell Line, Tumor
Centromeres
CHO Cells
Chromatin
Chromatin - genetics
Chromosomes
Chromosomes, Artificial, Human
Cricetinae
Cricetulus
Deoxyribonucleic acid
DNA
DNA, Satellite - genetics
epigenetics
Gene Expression
Gene Silencing
gene transfer
Genetic Vectors - genetics
Green Fluorescent Proteins - genetics
HeLa Cells
Humans
kinetochores
Life Sciences
Research Article
RNA, Transfer - genetics
transfer DNA
transfer RNA
Transgenes
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Summary: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, alphoidᵗᵉᵗᴼ-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 alphoidᵗᵉᵗᴼ-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.
ISSN:1420-682X
1420-9071
DOI:10.1007/s00018-013-1362-9