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Updated: May 11, 2026
10:28
Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers
Published on: September 20, 2018
Protecting a transgene expression from the HAC-based vector by different chromatin insulators.
Nicholas C O Lee1, Artem V Kononenko, Hee-Sheung Lee
1Laboratories of Molecular Pharmacology, National Cancer Institute, Bethesda, MD, 20892, USA.
Cellular and Molecular Life Sciences : CMLS
|May 17, 2013
Summary
Chromatin insulators are crucial for stable transgene expression within human artificial chromosomes (HACs). A tRNA gene (tDNA) insulator demonstrated the highest barrier activity, preventing epigenetic silencing in centrochromatin.
Area of Science:
- Epigenetics
- Molecular Biology
- Gene Therapy
Background:
- Human artificial chromosomes (HACs) are advanced vectors for gene delivery and expression.
- A novel HAC vector, alphoid(tetO)-HAC, features a conditional centromere with a gene-loading site in the centrochromatin domain.
- Long-term transgene expression within centrochromatin remains understudied.
Purpose of the Study:
- To evaluate the efficacy of three chromatin insulators (cHS4, gamma-satellite DNA, tDNA) in maintaining transgene expression within the centrochromatin of an alphoid(tetO)-HAC vector.
- To determine the impact of insulator function on preventing epigenetic silencing of transgenes located in close proximity to the centromere.
Main Methods:
- Insertion of an EGFP transgene into the alphoid(tetO)-HAC vector.
- Comparison of transgene expression levels in the presence of cHS4, gamma-satellite DNA, and tDNA insulators.
- Assessment of insulator activity in two different host cell lines over time.
Main Results:
- Insulator function was essential for stable EGFP transgene expression within the centrochromatin.
- The tDNA insulator, comprising two functional tRNA genes, exhibited the highest barrier activity against silencing.
- Genes lacking chromatin insulators were susceptible to epigenetic silencing due to their proximity to centrochromatin.
Conclusions:
- Chromatin insulators are necessary to prevent epigenetic silencing of transgenes within the centrochromatin of HACs.
- The tDNA insulator shows significant potential for enhancing the stability and expression of transgenes in HAC-based vectors.
- These findings highlight the importance of barrier elements for optimizing transgenesis using HAC technology.
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