5′ Transgenes drive leaky expression of 3′ transgenes in Cre-inducible bi-cistronic vectors

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-06-24 DOI:10.1016/j.omtm.2024.101288

Yasuyuki Osanai, Yao Lulu Xing, Shinya Mochizuki, Kenta Kobayashi, Jihane Homman-Ludiye, Amali Cooray, Jasmine Poh, Ayumu Inutsuka, Nobuhiko Ohno, Tobias D. Merson

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Abstract

Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences provide an invaluable tool for understanding the molecular regulation of cellular functions. The Cre-lox system is used for inducing the expression of recombinant proteins encoded within a bi-/poly-cistronic cassette. However, leak expression of transgenes is often observed in the absence of Cre recombinase activity, compromising the utility of this approach. To investigate the mechanism of leak expression, we generated Cre-inducible bi-cistronic vectors to monitor the expression of transgenes positioned either 5′ or 3′ of a 2A peptide or internal ribosomal entry site (IRES) sequence. Cells transfected with these bi-cistronic vectors exhibited Cre-independent leak expression specifically of transgenes positioned 3′ of the 2A peptide or IRES sequence. Similarly, AAV- vectors encoding bi-cistronic cassettes or fusion proteins revealed the selective Cre-independent leak expression of transgenes positioned at the 3′ end of the open reading frame. Our data demonstrate that 5′ transgenes confer promoter-like activity that drives the expression of 3′ transgenes. An additional STOP cassette between the 2A sequence and 3′ transgene dramatically decreased Cre-independent transgene expression. Our findings highlight the need for appropriate experimental controls when using Cre-inducible bi-/poly-cistronic constructs and inform improved design of vectors for more tightly regulated inducible transgene expression.

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5′ 转基因驱动 Cre 诱导的双组分载体中 3′ 转基因的泄漏表达

分子克隆技术能够同时表达两个或多个蛋白质编码序列，为了解细胞功能的分子调控提供了一种宝贵的工具。Cre-lox 系统用于诱导表达双/多核苷酸盒编码的重组蛋白。然而，在没有 Cre 重组酶活性的情况下，经常会观察到转基因的泄漏表达，从而影响了这种方法的实用性。为了研究泄漏表达的机制，我们生成了Cre诱导的双组词载体，以监测位于2A肽或内部核糖体进入位点（IRES）序列5′或3′的转基因的表达。用这些双组分载体转染的细胞表现出不依赖于 Cre 的泄漏表达，特别是位于 2A 肽或 IRES 序列 3′ 处的转基因。同样，编码双组分盒或融合蛋白的 AAV 载体也显示，位于开放阅读框 3′ 端的转基因具有选择性的不依赖 Cre 的泄漏表达。我们的数据证明，5′转基因具有类似启动子的活性，能驱动 3′转基因的表达。在 2A 序列和 3′ 转基因之间的附加 STOP 盒显著降低了 Cre 依赖性转基因的表达。我们的发现强调了在使用Cre诱导的双/多核苷酸构建体时进行适当实验控制的必要性，并为改进载体设计以实现更严格调控的诱导转基因表达提供了信息。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology

CiteScore

9.90

自引率

4.30%

发文量

163

审稿时长

12 weeks

期刊介绍： The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.