ArticleFrontiers in bioengineering and biotechnology2024
Large T antigen mediated target gene replication improves site-specific recombination efficiency.
Article in Frontiers in bioengineering and biotechnology, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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1 citing paper in PubMed.
- Development of a Lentiviral Vector for High-Yield Production of Synthetic and Recombinant GCase for Gaucher Disease Therapy.International journal of molecular sciences · 2025Article
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3 authors.
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Abstract
With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >10
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