High‐efficiency protein delivery into transfection‐recalcitrant cell types. Issue 3 (20th December 2019)

• Record Type: Journal Article
• Authors: Li, Zhenpeng; Cai, Zeqiong; Fu, Weixin; Liu, Ying; Tian, Chenglei; Wang, He; Fu, Tongtong; Wu, Zhenzhou; Wu, Donghai; Jin, Yongxin; Cheng, Zhihui; Terada, Naohiro; Liu, Lin; Wu, Weihui; Jin, Shouguang; Bai, Fang
• Is Part Of: Biotechnology and bioengineering. Volume 117:Issue 3(2020)
• Page Start: 816
• Page End: 831
• Publication Date: 2019-12-20
• Subjects: Cre/loxP -- pluripotent stem cells -- protein delivery -- transfection‐recalcitrant cells -- type III secretion system; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27245
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large‐scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac‐T3SS)‐mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self‐limiting growth. Compared to the method of transfection, the Bac‐T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard‐to‐transfect human cell lines. Furthermore, through the delivery of β‐lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac‐T3SS. This study provided an efficient and low‐cost proteofection strategy for laboratory use as well as for application in large‐scale cell manipulations. Abstract : A novel tool of intracellular protein delivery based on the Pseudomonas aeruginosa type III secretion system (T3SS) was developed in this study, which provides potential applications in efficient protein delivery for "transfection‐recalcitrant" cell types, such as epithelial, immune and pluripotent stem cells. By using this tool, Cre recombinase was produced by bacterial vehicle and delivered into target cells through T3SS, the translocated Cre mediated the loxP‐sites specific recombination both in vitro and in vivo.