PUBLICATION

p53 activation by knockdown technologies

Authors
Robu, M.E., Larson, J.D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S.A., and Ekker, S.C.
ID
ZDB-PUB-070614-15
Date
2007
Source
PLoS Genetics   3(5): e78 (Journal)
Registered Authors
Ekker, Stephen C., Farber, Steven, Larson, Jon D., Nasevicius, Aidas
Keywords
Embryos, Apoptosis, Cell death, Zebrafish, Phenotypes, Small interfering RNAs, Bright field microscopy, Microarrays
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*
PubMed
17530925 Full text @ PLoS Genet.
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.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping