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Related Experiment Video

Updated: Jun 26, 2025

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
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Large T antigen mediated target gene replication improves site-specific recombination efficiency.

Zening Wang1,2, Chuan Chen2, Xin Ge1,2

  • 1Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, United States.

Frontiers in Bioengineering and Biotechnology
|May 13, 2024
PubMed
Summary

SV40 large T antigen (TAg) expression boosts site-specific recombination (SSR) efficiency by amplifying donor DNA. This TAg-facilitated RMCE (T-RMCE) enables large-scale genomic screening and directed evolution in mammalian cells.

Keywords:
RMCEdirected evolutiongenome editinglarge T antigensite-specific recombinase

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Site-specific recombination (SSR) enables precise genomic modifications but is limited by low donor DNA availability.
  • SV40 large T antigen (TAg) is known to enhance DNA replication from SV40 origins.

Purpose of the Study:

  • To investigate if SV40 TAg expression can increase donor plasmid copy number and enhance SSR efficiency.
  • To develop a novel method for improving genomic engineering applications.

Main Methods:

  • Dual recombinase-mediated cassette exchange (RMCE) was performed in 293F cells.
  • Co-transfection with SV40 TAg was used to test its effect on SSR efficiency.
  • Donor plasmid amplification was assessed in correlation with recombination events.

Main Results:

  • TAg co-transfection significantly enhanced SSR efficiency in polyclonal cells.
  • A 12% RMCE efficiency was achieved in a monoclonal cell line, linked to donor plasmid amplification.
  • The T-RMCE system was used to construct libraries with >10^7 diversity.

Conclusions:

  • SV40 TAg expression effectively enhances SSR by amplifying donor DNA, overcoming a key limitation.
  • TAg-facilitated RMCE (T-RMCE) is a powerful tool for generating high-diversity libraries for directed evolution.
  • The principle of target gene amplification via TAg holds promise for broader genomic screening and gene editing applications.