Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors.
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Yanling
Y
The Crump Institute for Molecular Imaging, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
Iyer
Meera
M
Annala
Alexander J
AJ
Chappell
Steve
S
Mauro
Vincent
V
Gambhir
Sanjiv S
SS
D000818:Animals; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D008822:Mice, Transgenic; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D011877:Radionuclide Imaging; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D018398:Homeodomain Proteins; D009363:Neoplasm Proteins; C117745:Nkx6-2 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors