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5'转基因在Cre诱导型双顺反子载体中驱动3'转基因的渗漏表达。

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

作者信息

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

机构信息

Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia.

Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0431, Japan.

出版信息

Mol Ther Methods Clin Dev. 2024 Jun 24;32(3):101288. doi: 10.1016/j.omtm.2024.101288. eCollection 2024 Sep 12.

DOI:10.1016/j.omtm.2024.101288
PMID:39104576
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11298883/
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.

摘要

能够同时表达两个或更多蛋白质编码序列的分子克隆技术为理解细胞功能的分子调控提供了一个极其宝贵的工具。Cre-lox系统用于诱导双顺反子/多顺反子盒内编码的重组蛋白的表达。然而，在没有Cre重组酶活性的情况下，常常观察到转基因的渗漏表达，这损害了该方法的实用性。为了研究渗漏表达的机制，我们构建了Cre诱导型双顺反子载体，以监测位于2A肽或内部核糖体进入位点（IRES）序列5'或3'端的转基因的表达。用这些双顺反子载体转染的细胞表现出独立于Cre的渗漏表达，特别是位于2A肽或IRES序列3'端的转基因。同样，编码双顺反子盒或融合蛋白的腺相关病毒（AAV）载体也显示出位于开放阅读框3'端的转基因存在选择性的独立于Cre的渗漏表达。我们的数据表明，5'端转基因赋予启动子样活性，驱动3'端转基因的表达。在2A序列和3'端转基因之间添加一个额外的终止盒可显著降低独立于Cre的转基因表达。我们的研究结果强调了在使用Cre诱导型双顺反子/多顺反子构建体时需要适当的实验对照，并为更严格调控诱导型转基因表达的载体改进设计提供了参考。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/37db97abae8f/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/5a1d957e2d99/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/141c1a69700e/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/ec797d199fb9/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/e4b83b002abf/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/3f800b9c5eea/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/e414062d57ed/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/397d0fa0a726/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/37db97abae8f/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/5a1d957e2d99/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/141c1a69700e/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/ec797d199fb9/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/e4b83b002abf/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/3f800b9c5eea/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/e414062d57ed/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/397d0fa0a726/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e0c8/11298883/37db97abae8f/gr7.jpg

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