Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells

Felipe E Bravo,Jannel Acosta,Frank Camacho,Jorge R Toledo,Oliberto Sanchez,Alaín González,Natalie C Parra

doi:10.1038/s41598-020-69709-1

Abstract

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

Highlights

The generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events
The specific productivity (Qp) of mammalian cell lines usually correlates with the transgene messenger RNA (mRNA) levels, which depend on the transcriptional unit design, the transgene copy number integrated into the genome, and the chromatin arrangement at the integration s ite[4]
During the first stage of this work, we designed a strategy for the fluorescence-assisted sequential insertion of transgenes (FASIT)

Summary

Introduction

The generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. Transforming mammalian cell lines is often time-consuming and frequently generates low-producing clones with unstable gene e xpression[3] It is required high productivity and stability of the transgene expression for developing commercially viable processes. The generation of cell lines for recombinant protein production is done by two main strategies: the selection using dihydrofolate reductase (DHFR) and methotrexate (MTX)[5] or using glutamine synthetase (GS) and methionine sulfoximine (MSX)[6] For both systems, the selection of high-producing clones usually harbors transgenes organized in tandem arrays from tens to hundreds of copies. We used the sequential insertion of transgenes mediated by lentiviral vectors as a new method for generating stable clones producing high levels of a specific recombinant protein. Three whole rounds of transgene insertion-deletion were performed, showing a stepwise increasing of the hscFSH copy number together with the expression levels

Methods

Results

Conclusion