Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression

Rebecca Sizer; Richard Ingram; Bob White

doi:10.1002/biot.202400455

Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression

Rebecca Sizer, Richard Ingram, Bob White

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.

Original language	English
Article number	202400455
Number of pages	10
Journal	Biotechnology journal
Volume	20
DOIs	https://doi.org/10.1002/biot.202400455
Publication status	Published - 16 Feb 2025

Bibliographical note

Access to Document

10.1002/biot.202400455Licence: CC BY

Biotechnology Journal - 2025 - Sizer - Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous
© 2025 The Author(s).
Final published version, 1.32 MBLicence: CC BY

Cite this

@article{99336ecfa92b472a806eb1671172fd4b,

title = "Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression",

abstract = "Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.",

author = "Rebecca Sizer and Richard Ingram and Bob White",

note = "{\textcopyright} 2025 The Author(s).",

year = "2025",

month = feb,

day = "16",

doi = "10.1002/biot.202400455",

language = "English",

volume = "20",

journal = "Biotechnology journal",

issn = "1860-6768",

publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Barriers Composed of tRNA Genes Can Complement the Benefits of a Ubiquitous Chromatin Opening Element to Enhance Transgene Expression

AU - Sizer, Rebecca

AU - Ingram, Richard

AU - White, Bob

PY - 2025/2/16

Y1 - 2025/2/16

N2 - Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.

AB - Random integration of transgenes into host cell genomes often occurs in epigenetically unstable regions, leading to variable and unreliable transgene expression. To address this, biomanufacturing organizations frequently employ barrier elements, such as the widely-used ubiquitous chromatin opening element (UCOE). We have compared UCOE barrier activity against a barrier provided by tRNA genes. We demonstrate that the tRNA genes provide a more effective barrier than a UCOE in preventing transgene silencing in Chinese hamster ovary (CHO) cells. Nevertheless, the UCOE offers other benefits, increasing expression strongly, albeit transiently, and reducing production variability. Both the UCOE and tRNA genes counteract the repressive heterochromatin mark H3K9me3, but only the tRNA genes sustain euchromatic H3K27ac and recruitment of RNA polymerase II (Pol II) throughout long-term culture. A hybrid combining these distinct types of elements can provide benefits of both, enhancing expression in a more enduring manner. This synthetic hybrid offers potential for biomanufacturing applications.

U2 - 10.1002/biot.202400455

DO - 10.1002/biot.202400455

M3 - Article

SN - 1860-6768

VL - 20

JO - Biotechnology journal

JF - Biotechnology journal

M1 - 202400455

ER -