World Moleuclar Imaging Congress location:Nice, France date:11-13 September 2008
Aims: Reporter gene imaging for gene and cell therapy often requires expression of multiple transgenes by the same construct. Different strategies have been used but systematic comparison is lacking. Here, we report a comparison of the currently available approaches (including internal ribosomal entry sites (IRES), 2A-like peptides, and a bidirectional promoter) that allow expression of multiple reporter genes (i.e. green fluorescent protein (eGFP), firefly luciferase (fLuc), Herpes simplex virus thymidine kinase (HSV-tTK)) in a lentiviral vector context, both in cell culture and in rodent brain.
Methods: LV transfer plasmids were constructed with both eGFP and fLuc linked by the EMCV IRES, four different self cleaving 2A-like sequences, a 10-fold repeat of a 9-nucleotide cellular sequence with IRES-activity (10CI), as a fusion protein and using a bidirectional promoter. eGFP and fLuc expression after LV transduction was verified by fluorescence microscopy, western blot (WB), FACS and fLuc assay. In addition, LVs were stereotactically injected into the mouse striatum. Expression was measured by BLI and stereological quantification of eGFP-positive volume after eGFP immunohistochemistry. Using two different 2A-sequences, a multicistronic LV expressing eGFP, fLuc and HSV-tTK was constructed. Reporter gene expression was tested by fLuc assay, FACS analysis and a suicide gene assay in cellulo.
Results: eGFP and fLuc expression in transduced cells was obtained from all constructs. WB confirmed expression of both reporters and showed predominantly cleaved protein for most 2A-like peptides. Expression of eGFP and fLuc reporters was high in cellulo and in vivo for the constructs containing IRES and 2A-sequences as compared to the other constructs. Efficient eGFP, fLuc and HSV-tTK expression was shown in transduced cells with a triple reporter lentivector using two distinct 2A-like sequences.
Conclusion: The EMCV IRES and self-cleaving 2A-like peptides are most suitable for efficient coexpression of multiple imaging reporters in rodent brain using multicistronic lentivectors.