Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.
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.
Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Flanders, Belgium.
Abdelilah
A
Ibrahimi
Greetje
G
Vande Velde
Veerle
V
Reumers
Jaan
J
Toelen
Irina
I
Thiry
Caroline
C
Vandeputte
Sofie
S
Vets
Christophe
C
Deroose
Guy
G
Bormans
Veerle
V
Baekelandt
Zeger
Z
Debyser
Rik
R
Gijsbers
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015774:Ganciclovir / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014161:Transduction, Genetic
D010455:Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015774:Ganciclovir