The application of transgenic systems is of great importance in both basic research for studying protein functions and in clinical gene therapy. Regulated expression systems are necessary top recisely influence complex cellular processes. In this study, we present a system that enables defined protein expression by utilizing the properties of internal ribosome entry site (IRES),which reduces downstream protein expression more effectively than 2A sequences. Lentivector donor sequences were inserted into the last intron of the highly expressed HSPA8gene using the HITI method (Homology-Independent Targeted Integration). These donors contain a splice acceptor site followed by the last exon of HSPA8, which is linked to them Cherry gene either via IRES or 2A-coding sequences. For the selection of transduced cells containing the insert, a puromycin resistance gene expressed either from a tricistronic mRNA(with a 2A sequence) or from an exogenous EF1α promoter was used. Analysis by flow cytometry (FACS) and fluorescence microscopy showed that the EF1αpromoter before the puromycin resistance gene reduced the mCherry expression and led to insufficient selection. In contrast, the donors with the 2A sequence before the puromycin resistance gene lead to a satisfactory selection. The donors with 2A linker connecting HSPA8and mCherry exhibited stronger mCherry expression compared to IRES-containing vectors, confirming the desired defined expression patterns. In summary, two expression systems were developed, achieving two distinct stable expressions of the protein of interest.
Andrea Schmidhofer (Fri,) studied this question.