comparison of ires and f2a-based locus-specific multicistronic expression in stable mouse lines比较的忿怒和f2a-based locus-specific multicistronic表达稳定的鼠标线.pdfVIP

Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines . . Hsiao Yun Chan , Sivakamasundari V. , Xing Xing, Petra Kraus, Sook Peng Yap, Patricia Ng, Siew Lan Lim, Thomas Lufkin* Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, Singapore Abstract Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9- EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP