Strategies for all-at-once and stepwise selection of cells with multiple genetic manipulations.

Mei Horikawa, Hisataka Sabe, Yasuhito Onodera

Biochemical and biophysical research communications 2021 Dec 10

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The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology. Copyright © 2021 Elsevier Inc. All rights reserved.

Citation

Mei Horikawa, Hisataka Sabe, Yasuhito Onodera. Strategies for all-at-once and stepwise selection of cells with multiple genetic manipulations. Biochemical and biophysical research communications. 2021 Dec 10;582:93-99