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Generation of stable cell lines using readthrough expression from lentiviral integration.

Anish Jadav1, Kevin Truong2,3

  • 1Institute of Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON, M5S 3G9, Canada.

Biotechnology Letters
|September 17, 2021
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Summary

Readthrough expression, typically unwanted in lentiviral gene delivery, can be harnessed to express functional proteins. This study demonstrates its utility for creating stable cell lines and potentially expressing larger genes.

Keywords:
Cell-based therapyGene expressionLentivirusPromoterReadthrough

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Area of Science:

  • Molecular Biology
  • Gene Therapy
  • Cell Biology

Background:

  • Lentiviral vectors are common tools for stable gene integration and transgene expression.
  • Unintended readthrough expression from endogenous promoters can lead to non-functional protein products.

Purpose of the Study:

  • To demonstrate that lentiviral readthrough expression can be a beneficial phenomenon.
  • To show readthrough expression can reduce lentiviral transfer plasmid size.
  • To generate functional proteins using readthrough expression.

Main Methods:

  • Utilized lentiviral vectors to induce readthrough expression in HEK293 cells.
  • Generated stable cell lines expressing fluorescent reporters, antibiotic resistance fusion proteins, and vascular endothelial growth factor receptor 2.
  • Assessed protein functionality through fluorescence, antibiotic resistance assays, and receptor-mediated signaling.

Main Results:

  • Successfully generated HEK293 cell lines exhibiting functional protein expression via readthrough.
  • Demonstrated functionality of fluorescent reporters, antibiotic resistance markers, and VEGFR2.
  • Confirmed readthrough expression as a viable method for producing functional proteins.

Conclusions:

  • Readthrough expression can be a desirable mechanism for lentiviral gene delivery.
  • This approach enables the expression of functional proteins and reduces plasmid size.
  • Suggests potential applications in expressing larger genes or genetic circuits, such as in cell-based therapeutics.