Mouse adenovirus (MAV-1) expression in primary human endothelial cells and generation of a full-length infectious plasmid.
Using RT-PCR, we show that mouse adenovirus type I (MAV-1) is capable of infecting and expressing in various cell types, specifically human endothelial cells. The capability of MAV-1 to infect and express in human endothelial cells makes it a potentially useful alternative to the use of human adenoviruses type 2/5 (Ad2/5) in virus-based gene therapy, although presently MAV-1 can only be produced at lower titers than Ad2/5. In this report, we present methods for the purification of MAV-1 DNA and use of this DNA along with a modified bacteria-based homologous recombination protocol to generate a full-length plasmid clone of MAV-1 DNA. Using various transfection procedures, we show that this plasmid MAV-1 DNA can generate plaques of MAV-1 virus, albeit at low efficiencies (about 0. 2 p.f.u./microg DNA). Furthermore, the construction of an MAV-1 plasmid along with its capability to express in human cells justifies the full development of MAV-1 into a system of gene therapy.
Nguyen
T
T
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
Nery
J
J
Joseph
S
S
Rocha
C
C
Carney
G
G
Spindler
K
K
Villarreal
L
L
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004727:Endothelium / Q000166:cytology / Q000821:virology*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D010957:Plasmids; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004247:DNA