Monitoring adenoviral p53 transduction efficiency by yeast functional assay

M. Tada, S. Sakuma, R. D. Iggo, H. Saya, Y. Sawamura, T. Fujiwara, J. A. Roth

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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 (τ(o) = [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 τ(o) 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.

Original languageEnglish
Pages (from-to)339-344
Number of pages6
JournalGene Therapy
Volume5
Issue number3
DOIs
Publication statusPublished - 1998 Jan 1
Externally publishedYes

Keywords

  • Adenovirus
  • Transcripts
  • Transduction efficiency
  • Yeast p53 functional assay
  • p53

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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  • Cite this

    Tada, M., Sakuma, S., Iggo, R. D., Saya, H., Sawamura, Y., Fujiwara, T., & Roth, J. A. (1998). Monitoring adenoviral p53 transduction efficiency by yeast functional assay. Gene Therapy, 5(3), 339-344. https://doi.org/10.1038/sj.gt.3300605