Abstract
1139
Objectives: Reporter gene imaging for gene and cell therapy often requires expression of multiple transgenes by the same construct, to monitor expression of a gene of interest or for multimodality detection. Different strategies have been used but systematic comparison is lacking. We aimed to compare these strategies for lentiviral vector (LV) applications with bioluminescence imaging (BLI). Methods: LV transfer plasmids were constructed with both green fluorescent protein (eGFP) and firefly luciferase (FLuc) alone or linked by the EMCV internal ribosome entry site (IRES), two different self cleaving 2A sequences (F2A: picornavirus-derived (FMDV), T2A: insect virus derived (Thosea asigna)), a 10-fold repeat of a 9-nucleotide cellular sequence with IRES-activity (9ntCI) and as a fusion protein (Fu). In all constructs eGFP is present in the first position. We also used a bidirectional promoter in a different transfer plasmid (BiD). eGFP and FLuc expression after plasmid transfection or LV transduction was verified by fluorescence microscopy, western blot, FACS and BLI. In addition,LV were stereotactically injected into the mouse striatum (n=3). Expression was measured by BLI and stereological determination of eGFP-positive volume after eGFP immunohistochemistry. Results: Intense eGFP expression was obtained from all constructs. WB confirmed expression of both eGFP and Fluc but showed a predominantly non-cleaved protein for F2A. Transfection showed relatively similar eGFP levels by all constructs with Fu as lowest. Fluc activities were within the same order of magnitude except for 9ntCI and BiD whose values were more than 1 log lower. Transduction with LV showed similar results except for low eGFP values in 9ntCI. In vivo measurements showed highest Fluc activity values for T2A and Fluc, followed by Fu. The values for 9ntCI and BiD were low. Conclusions: Self-cleaving 2A sequences appear to be a good choice for LV bicistronic constructs, but different sequences need to be tested to assure adequate cleavage. The other strategies performed less well in the context of our LV system.
- Society of Nuclear Medicine, Inc.