Horikawa Mei, Sabe Hisataka, Onodera Yasuhito
Department of Molecular Biology, Faculty of Mecidine and Graduate School of Medicine, Hokkaido University, Sapporo, 060-8638, Japan.
Department of Molecular Biology, Faculty of Mecidine and Graduate School of Medicine, Hokkaido University, Sapporo, 060-8638, Japan; Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido University, Sapporo, 060-8638, Japan.
Biochem Biophys Res Commun. 2021 Dec 10;582:93-99. doi: 10.1016/j.bbrc.2021.10.016. Epub 2021 Oct 8.
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.
细胞的基因操作及其后续筛选对于细胞生物学研究而言不可或缺。尽管抗生素抗性基因常被用作筛选标记，但每种选择剂的条件优化仍有必要。在此，我们利用分裂内含肽和药物可选择标记嘌呤霉素N - 乙酰转移酶（PAC）开发了一种系统，该系统仅使用嘌呤霉素就能实现对同时或先后转染多个基因构建体的细胞进行筛选。活性PAC酶通过内含肽介导的反式剪接在几个天然或工程化的丝氨酸/半胱氨酸残基处得以重构。通过基于细胞对不同嘌呤霉素浓度的耐受性选择最佳切割位点，可轻松实现活性PAC的多次切割和重构。为实现逐步筛选方法，使用由HIV - 1 gag - pol和VSV - G组成的病毒样颗粒（VLP）将PAC - 内含肽片段转导至细胞中。PAC - 内含肽 - VLP成功赋予了足够的PAC活性用于嘌呤霉素筛选，在无VLP时该活性会迅速降低。我们的研究结果展示了一种通用策略，可用于建立用于一次性或逐步筛选多种基因操作的标记，这将在许多生物学领域中发挥作用。
