pVIVO-Lucia/SEAP

• haGRP78 and hGRP94 prom: The hamster GRP78 and human
GRP94 promoters drive weak levels of expression in normal conditions and are induced in stress conditions prevailing inside tumors, such as glucose deprivation and hypoxia. Within the tumor micro-environment, the GRP promoters yield persistent expression whereas the activity of viral promoters declines rapidly.

• hFerH and hFerL composite promoters: Ferritin is a 24 subunit protein composed of two subunit types, termed H (heavy) and L (light). Ferritin is ubiquitously expressed. To eliminate the iron regulation of the ferritin promoters, the 5’UTR of FerH and FerL have been replaced by the 5’UTR of the mouse and chimpanzee elongation factor 1 (EF1) genes, respectively.

• SV40 enhancer which is comprised of a 72-base-pair repeat allows the enhancement of gene expression in a large host range. The enhancement varies from 2-fold in non-permissive cells to 20-fold in permissive cells. Furthermore, the SV40 enhancer is able to direct nuclear localization of plasmids.

• CMV enhancer: The major immediate early enhancer of the human cytomegalovirus (HCMV), is composed of unique and repeated sequence motifs. The HCMV enhancer can substitute for the 72-bp repeats of SV40 and is severalfold more active than the SV40 enhancer.

• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.

• pMB1 ori: a minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori.

• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.

• Hygro-ΔCpG is a new allele of the hph gene conferring resistance to hygromycin B. In order to reduce the immunogenicity of this bacterial gene all CpG motifs have been removed by chemically synthesizing the gene. The Hygro-ΔCpG gene allows the selection of E. coli clones transformed with a pVIVO plasmid.
Note: Stable transfection of mammalian clones cannot be performed due to the absence of a eukaryotic promoter upstream of the Hygro-ΔCpG gene.

• Term: The E. coli rps O terminator allows efficient transcription termination of the Hygro-ΔCpG gene.

• EF1 pAn is a strong polyadenylation signal.

• SEAP is a secreted form of human embryonic alkaline phosphatase. Unlike endogenous alkaline phosphatases, SEAP is extremely heat stable and resistant to the inhibitor L-homoarginine. It catalyses the hydrolysis of pNitrophenyl phosphate (pNpp) producing a yellow end product. SEAP expression can be readily quantified by collecting samples of culture medium and measuring the hydrolysis of pNpp with a  spectrophometer at 405 nm. 

• Lucia luciferase is a synthetic CpG-free gene that codes for a secreted coelenterazine-utilizing luciferase. ORF size (from the ATG to the stop codon): 634 bp. Lucia luciferase activity can be evaluated using QUANTI-Luc™, an assay reagent containing all the components required to quantitively measure the activity of coelenterazine-utilizing luciferases.