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bioRxiv · 10.1101/2024.08.26.609662

Rapid selection of transgenic mammalian cells via diphtheria toxin resistance

Scherer, D.·Honrath, S.·Leroux, J.-C.·Burger, M.
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Abstract

The ability to generate stable transgenic mammalian cell lines is crucial to the investigation of gene functions and the production of recombinant proteins. Currently, mammalian cells can be readily transfected in cell culture settings via both viral and nonviral vectors to induce transgene expression. However, there is an unmet need for efficient selection of transfected cells, since current methods involve rather inefficient antibiotic selection protocols or require the coexpression of fluorescent marker proteins, followed by laborious cell sorting procedures. Our aim was to implement a rapid and efficient in situ selection approach for transgene-expressing human cells, using an engineered diphtheria toxin (DT) resistance-based selection, referred to as selecDT. We demonstrated that selecDT is expressed on the surface of modified human cells, provides efficient protection from DT by inactivating its uptake receptor, and, therefore, enables selection. Current antibiotic-based methods require selection periods of more than a week and often achieve only limited cell enrichment. With selecDT, one day of selection is sufficient to obtain nearly 100% enrichment. The DT resistance described herein may thus positively impact biotechnological processes and biomedical research.

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Scherer, D., Honrath, S., Leroux, J.-C., Burger, M.. 2024-08-26. Rapid selection of transgenic mammalian cells via diphtheria toxin resistance. https://doi.org/10.1101/2024.08.26.609662

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