MONITORING ADENOVIRAL P53 TRANSDUCTION EFFICIENCY BY YEAST FUNCTIONALASSAY

Monitoring the transduction efficiency is of paramount importance in g ene therapy. To monitor adenovirus-mediated wild-type p53 gene transfe r, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cel lular and viral p53 transcripts followed by quantitive assessment of m utant 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 r atio of endogenous p53 mRNA (P2), and other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection o f cell lines homozygous for mutant p53, the apparent transduction effi ciency calculated (tau(0)=[P1-P2]/[1+P2]) correlated with the level of P21 expression. Transduction efficiency in heterozygous wild-type/mut ant HUG31 cells increased linearly with multiplicity of infection (MOI ) for tau(0) value, in keeping with theoretical predictions. These res ults suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.