Improvement of reporter activity by IRES-mediated polycistronic reporter system.
Many in vitro and in vivo applications for transgenesis require co-expression of heterologous genes. The use of internal ribosome entry sites (IRESs) in dicistronic expression vectors enables the expression of two genes controlled by one promoter in target cells or whole organisms. Here we describe the expansion of IRES exploitation to generate multicistronic vectors capable of expressing multiple reporter genes, especially to improve the fluorescence yield of autofluorescent reporter gene products such as green fluorescent protein (GFP). We found that the increase in fluorescence output of GFP is proportional to the number of IRES-GFP repeats in the multicistronic vector. At least four genes can be expressed from a multicistonic vector by using tandem IRES elements, with no significant alteration of the expression level of the cap-dependent translated gene. Moreover, gene expression under the control of multiple IRES element has no effect on the posttranscriptional regulation through 3'-untranslated regions (3'UTR). Thus, endogenous gene expression and regulation, especially those controlled by weak promoters, can be analyzed with our IRES-dependent polycistronic reporter gene expression system.
Bouabe
Hicham
H
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, 80336 Munich, Germany. bouabe@mvp.uni-muenchen.de
Fässler
Reinhard
R
Heesemann
Jürgen
J
D020413:3' Untranslated Regions / Q000737:chemistry; D002460:Cell Line; D005456:Fluorescent Dyes / Q000032:analysis; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D038621:Regulatory Sequences, Ribonucleic Acid
D020413:3' Untranslated Regions; D005456:Fluorescent Dyes; D012315:RNA Caps; D038621:Regulatory Sequences, Ribonucleic Acid; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins