title: p53 activation by knockdown technologies.
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.
authors.affiliation: University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Mara E
authors.initials: ME
authors.lastname: Robu
authors.forename: Jon D
authors.initials: JD
authors.lastname: Larson
authors.forename: Aidas
authors.initials: A
authors.lastname: Nasevicius
authors.forename: Soraya
authors.initials: S
authors.lastname: Beiraghi
authors.forename: Charles
authors.initials: C
authors.lastname: Brenner
authors.forename: Steven A
authors.initials: SA
authors.lastname: Farber
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ekker
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016477:Artifacts; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005821:Genetic Techniques; D009025:Morpholines / Q000494:pharmacology; D060172:Morpholinos; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010641:Phenotype; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D009025:Morpholines; D060172:Morpholinos; D020033:Protein Isoforms; D016159:Tumor Suppressor Protein p53