p53 Activation by Knockdown Technologies

PLoS Genet. 2007 May 25;3(5):e78. doi: 10.1371/journal.pgen.0030078. Epub 2007 Apr 10.

Abstract

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Artifacts
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Embryo, Nonmammalian / cytology
  • Embryo, Nonmammalian / drug effects
  • Embryo, Nonmammalian / embryology
  • Embryo, Nonmammalian / metabolism
  • Embryonic Development / drug effects
  • Embryonic Development / genetics
  • Gene Expression Regulation, Developmental / drug effects
  • Genetic Techniques*
  • Morpholines / pharmacology
  • Morpholinos
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism
  • Phenotype
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Sensitivity and Specificity
  • Substrate Specificity
  • Time Factors
  • Transcriptional Activation*
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics*
  • Tumor Suppressor Protein p53 / metabolism
  • Zebrafish / embryology
  • Zebrafish / genetics*

Substances

  • Cyclin-Dependent Kinase Inhibitor p21
  • Morpholines
  • Morpholinos
  • Protein Isoforms
  • Tumor Suppressor Protein p53