Development of high-throughput functional screening of therapeutic genes, using a hemagglutinating virus of Japan envelope vector.
Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.
Nishikawa
Tomoyuki
T
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
Nakagami
Hironori
H
Matsuki
Atsushi
A
Maeda
Akito
A
Yo
Chiah Yian
CY
Harada
Taku
T
Morishita
Ryuichi
R
Tamai
Katsuto
K
Kaneda
Yasufumi
Y
D002460:Cell Line; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004730:Endothelium, Vascular / Q000502:physiology; D015723:Gene Library; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D029082:Sendai virus
D018076:DNA, Complementary