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• W4280491119 abstract "Abstract Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences in a cell type of interest provide an invaluable tool for understanding the molecular regulation of cellular functions. DNA recombination employing the Cre-lox system is commonly used as a molecular switch for inducing the expression of recombinant proteins encoded within a bicistronic cassette. In such an approach, the two protein-coding sequences are separated by a 2A peptide or internal ribosome entry site (IRES), and expression is designed to be strictly Cre-dependent by using a lox -STOP- lox cassette or flip-excision (FLEX) switch. However, low-level or ‘leaky’ expression of recombinant proteins is often observed in the absence of Cre activity, potentially compromising the utility of this approach. To investigate the mechanism of leaky gene expression, we generated pCAG-lox-GFP-STOP-lox-Transgene A-2A-Transgene B vectors, which are designed to express nuclear-targeted GFP in the absence of Cre, and express both transgenes A and B after Cre-mediated recombination. We found that cells transfected with these bicistronic vectors exhibited low-level Cre-independent expression specifically of the transgene positioned 3′ of the 2A peptide. We observed similar results in vivo by viral transduction of the adult mouse cerebral cortex with AAV-mutagenesis of putative transcription factor binding sites that the 5′ transgene confers promoter-like activity that drives expression of the 3′ transgene. Finally, we demonstrate that inclusion of an additional lox-STOP-lox cassette between the 2A sequence and 3′ transgene dramatically reduces the extent of Cre-independent leaky gene expression. Our findings highlight that caution should be applied to the use of Cre-dependent bicistronic constructs when tight regulation of transgene expression is desired and provide a guide to preventing leaky gene expression when the expression of more than one protein is required." @default.
• W4280491119 created "2022-05-22" @default.
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• W4280491119 date "2022-04-23" @default.
• W4280491119 modified "2023-10-16" @default.
• W4280491119 title "5′ transgenes drive leaky expression of 3′ transgenes in inducible bicistronic vectors" @default.
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• W4280491119 doi "https://doi.org/10.1101/2022.04.23.489261" @default.
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