1. Volume 12, Issue 3, Pages 569-574 (September 2005)
A picornaviral 2A-like sequence-based tricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reporter system 
Mark J. Osborn, Angela Panoskaltsis-Mortari, Ron T. McElmurry, Scott K. Bell, Dario A.A. Vignali, Martin D. Ryan, Andrew C. Wilber, R. Scott McIvor, Jakub Tolar, Bruce R. Blazar 
Molecular Therapy 
Volume 12, Issue 3, Pages (September 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Tricistronic vector constructs. (A) Schematic of the cleavable pLB construct containing the IDUA downstream of the CMV promoter in the pCDNA3.1D TOPO vector (Invitrogen). Subsequently the P2A–luciferase–T2A–DsRed2 fragment, generated by recombinant PCR as described by Szymczak [8] and Higuchi [26], was cloned in-frame, downstream of the IDUA gene, resulting in the plasmid pLB. The IDUA cDNA was a gift from E. Neufeld [27], luciferase was derived from the pGL-3 vector (Promega), and DsRed2 was subcloned from the pIRES-DsRed2 vector (Clontech). Arrow indicates the cleavage sight between the 2A glycine and the 2B proline. (B) Individual cleavage products with associated 2A-like sequences at the C (IDUA and luciferase) and/or N (luciferase and DsRed2) terminus of the protein products. (C) The P2A sequence was mutated from PGP to AVP or (D) completely deleted, which should decrease the cleavage efficiency between IDUA and luciferase [13]. The T2A sequence was maintained to monitor transfection efficiency by DsRed2 expression.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
In vitro analysis of IDUA, luciferase, and DsRed2 gene expression in NIH 3T3 cells. Endotoxin-free plasmid (Qiagen) was used to transfect 106 3T3 cells using the Effectene transfection reagent (Qiagen) and monitoring for gene expression 48 h posttransfection. (A) In vitro IDUA activity. Cellular supernatant was removed and assayed in triplicate for IDUA activity using 4-methylumbelliferyl α-l-iduronide as the substrate as described [22]. Controls include an equal number of untransfected 3T3s and 106 BM cells. p2A-M represents cells transfected with the mutated (PGP → AVP) plasmid, and –PGP represents cells transfected with the P2A PGP-deleted mutant plasmid. IDUA activity was determined using a standard curve of known amounts of 4-methylumbelliferone (Sigma). Values are presented as IDUA/nmol/μg of protein/4 h and standard deviation is shown. (B) In vitro luciferase activity. Cells transfected with the cleavable (pLB), uncleavable p2A-M, or uncleavable –PGP plasmid were harvested and lysed in 1× Reporter Lysis Buffer (Promega) and seeded in triplicate in flat-bottom plates followed by addition of Luciferase Assay Buffer (Promega) and photon emission was measured for 5 s using a Chameleon 425–100 multilabel counter (Hidex). Cell lysate protein content was assessed using the Proteostain Protein Quantification Kit (Dojindo Molecular Technologies) and luciferase activity is expressed as relative light units (RLU)/mg of protein ± SD. (C) In vitro DsRed2 expression. 3T3 cells 48 h posttransfection (or untransfected cells as a control) were trypsinized and analyzed on a FACSCalibur (Becton-Dickinson) for DsRed2 activity. (D) Western blot analysis of cleavage activity. Antibody specificity for luciferase was determined using recombinant luciferase (lane 1). Protein concentration was determined from cell lysates using the Proteostain Protein Quantification Kit (Dojindo Molecular Technologies). Equal amounts of protein (50 μg) from pLB- (lane 2), p2A-M- (lane 3), or –PGP- (lane 4) transfected or untransfected (not shown) cells were separated on a NuPAGE 10% Bis-Tris gel (Invitrogen), transferred to a PVDF membrane (Invitrogen), and probed with a biotinylated polyclonal anti-luciferase antibody (Abcam), followed by addition of streptavidin-conjugated HRP (Abcam), and then developed using the ECL Western Blotting Analysis System (Amersham Biosciences). Cleaved and uncleaved luciferase is indicated by labeled arrows.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Tricistronic versus monocistronic vector gene expression. NOD-SCID mice were infused by hydrodynamic tail vein injection with equimolar amounts (50 μg) of pLB, monocistronic IDUA (35 μg), or monocistronic luciferase (37 μg) as described [23] and analyzed at 48 h postinjection. (A) In vivo IDUA activity. Serum was harvested and IDUA activity was assessed as before [22]. Animals receiving monocistronic luciferase served as controls. Duplicate experiments with three animals each were used and SEM is shown. (B) Whole-body in vivo luciferase imaging. Animals were anesthetized and administered 150 μg/g d-luciferin (Xenogen) by intraperitoneal injection and imaged for luciferase activity using the Xenogen IVIS imaging system. The scale bar on the right represents photon emission as photons/s/cm2/sr. Representative animals of tricistronic plasmid-injected (animal 1), control (IDUA injected, animal 2), and monocistronic luciferase-injected (animal 3) recipients are shown. (C) Comparison of luciferase activity between mono- and tricistronic-injected animals. A region of interest (ROI; red circle in B) was placed around the area of luciferase expression and the number of photons/s/cm2 was determined within the ROI using LivingImage software version 2.50 (Xenogen). Graphical analysis used the mean of photons counted in the ROI of four animals for each group over exposure times of 0.1–90 s with SEM shown. (D) Whole-organ DsRed2 expression. Animals were anesthetized and the liver was exposed following midline incision and imaged for DsRed2 expression using a Retiga EXi Fast 1394 camera (Q Imaging) mounted to a Leica MZFLII stereomicroscope as described [24]. No fluorescence was observed in uninjected or control plasmid-receiving animals (data not shown due to the fact that the background is nil). (E) Cellular DsRed2 expression. A region of liver, determined by whole-organ imaging to be expressing DsRed2, was dissected and homogenized so that a cytospin could be performed. DAPI nuclear staining (Molecular Probes) was performed followed by microscopy analysis using a BX51 fluorescence microscope (Olympus). Cells expressing DsRed2 are on the top, the DAPI image is in the middle, and a merged image showing DsRed2-positive hepatocytes with nuclei is shown at the bottom.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions