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

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-FLEX 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 lox-STOP-lox 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.

Keywords

• gene expression
• bi-cistronic vector
• poly-cistronic vector
• leak expression
• FLEx
• DIO