Article

Wang, Y, Iyer, M, Annala, AJ, Chappell, S, Mauro, V and Gambhir, SS. Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors. J Nucl Med 46: 667-674

The Crump Institute for Molecular Imaging, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
Journal of Nuclear Medicine (Impact Factor: 5.56). 05/2005; 46(4):667-74.
Source: PubMed

ABSTRACT Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo.
We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET.
In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES.
These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

0 Followers
 · 
84 Views
  • Source
    • "The presence and functionality of fructose transporters in human breast cancer cells may have diagnostic and therapeutic implications [143]. Wang et al. [144] showed that 4-bromo-2-{18F}fluoro-l- phenylalanine-fructose (L-{18F}FBPA-Fr) uptake is superior to that of {18F}FDG in microPET brain tumor imaging studies. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cancer cells, as with most mammalian cells, depend on a continuous supply of glucose; not only as a precursor of glycoproteins, triglycerides and glycogen, but also as an important source of energy. This review concentrates on GLUT transporter expression in both normal and cancerous classical sex-steroid hormone tissues (i.e. breast, uterus, ovary, testis and prostate, among others). Given the importance of estrogen, progesterone and androgens in carcinogenesis, as well as in survival and propagation of these cancers, this review also highlights the current literature on hormone regulation of glucose transporters and on the role of hypoxia in their expression. Given the recent explosion of information on the newer GLUT6-12 family members, a brief overview on their function and general expression has been included. Finally, an insight into the use of glucose transporters as markers of cancer progression and clinical outcome is also discussed.
    Current Vascular Pharmacology 11/2009; 7(4):534-48. DOI:10.2174/157016109789043928 · 2.91 Impact Factor
  • Source
    • "Nevertheless, this construct is suited for applications in which low expression levels are desirable, as suggested in the original publication (Amendola et al., 2005). To enhance reporter expression downstream of the IRES element, we attempted to use LV-10CI, reported to possess IRES activity and to lead to 12-fold greater reporter activity when compared with the EMCV IRES (Wang et al., 2005). In the present work, LV-10CI mediated low eGFP and fLuc expression levels in various cell types, barely above the background signal. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene discovery and gene therapy call for advanced technologies to reliably assess gene expression; efficient coupling of gene expression to the expression of reporter genes is critical. Various noninvasive molecular imaging modalities have emerged to track biological processes in animal models. Here, we evaluate various strategies to link transgene expression with that of an (imaging) reporter gene. Using lentiviral vectors containing internal ribosomal entry sites (IRES), 2A-like peptides, or a bidirectional promoter, we compared their ability to ensure efficient coexpression of multiple reporter genes. Although the encephalomyocarditis virus (EMCV) IRES yielded functional bicistronic vectors, the expression level of the reporter downstream of IRES was consistently lower than that of the upstream transgene. Interestingly, peptide 2A constructs performed best in vitro and in vivo, providing effective noninvasive follow-up of transgene expression and having reporter gene expression levels in line with that of the single reporter constructs. The intrinsic "cleavage" property of the peptide 2A sequences allows each protein to be produced at proportional levels, opening ample possibilities for functional genomics and future gene therapeutic applications. Last, using various peptide 2A sequences, we engineered the triple reporter LV-3R (i.e., eGFP, fLuc, HSV1-sr39tk), enabling efficient multimodality readouts in vivo.
    Human gene therapy 06/2009; 20(8):845-60. DOI:10.1089/hum.2008.188 · 3.62 Impact Factor
  • Source
    • "Although these in vitro systems are available and easy to perform, they suffer from the common problem that many tissue specific transcription factors are lost when cells are kept in an artificial environment for an extended period of time. Now we have constructed transient mouse model by hydrodynamic gene transfer to examine the activity of promoters and the effect of the HBV enhancers on the activity of the promoters in whole animals under true physiological conditions by the IVIS camera, which provides quantitative bioluminescence imaging of live mice [10] [11] [12]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.
    FEBS Letters 10/2008; 582(23-24):3552-6. DOI:10.1016/j.febslet.2008.09.035 · 3.34 Impact Factor
Show more