A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.
Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.
Urabe
M
M
Department of Molecular Biology, Institute of Hematology, Jichi Medical School, Tochigi, Japan.
Hasumi
Y
Y
Ogasawara
Y
Y
Matsushita
T
T
Kamoshita
N
N
Nomoto
A
A
Colosi
P
P
Kurtzman
G J
GJ
Tobita
K
K
Ozawa
K
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005814:Genes, Viral; D005819:Genetic Markers; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics*
D017931:DNA Primers; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D008156:Luciferases