Monitoring adenoviral p53 transduction efficiency by yeast functional assay.
Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = [P1-P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.
Tada
M
M
Department of Neurosurgery, Hokkaido University School of Medicine, Sapporo, Japan.
Sakuma
S
S
Iggo
R D
RD
Saya
H
H
Sawamura
Y
Y
Fujiwara
T
T
Roth
J A
JA
D000256:Adenoviridae / Q000235:genetics*; D015152:Blotting, Northern; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D015870:Gene Expression; D016158:Genes, p53; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53