p53 activation by knockdown technologies

Mara E Robu; Jon D Larson; Aidas Nasevicius; Soraya Beiraghi; Charles Brenner; Steven A Farber; Stephen C Ekker

doi:10.1371/journal.pgen.0030078

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p53 activation by knockdown technologies

Journal article

Open access

Peer reviewed

p53 activation by knockdown technologies

Mara E Robu, Jon D Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner, Steven A Farber and Stephen C Ekker

PLoS genetics, Vol.3(5), pp.787-801

05/25/2007

DOI: 10.1371/journal.pgen.0030078

PMCID: PMC1877875

PMID: 17530925

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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.

Phenotype

Embryo, Nonmammalian - cytology

Apoptosis - drug effects

Transcriptional Activation

Embryo, Nonmammalian - metabolism

Substrate Specificity

Embryo, Nonmammalian - embryology

Neurons - cytology

Zebrafish - embryology

Embryo, Nonmammalian - drug effects

Tumor Suppressor Protein p53 - genetics

Cyclin-Dependent Kinase Inhibitor p21 - genetics

Protein Isoforms - metabolism

Time Factors

Sensitivity and Specificity

Cyclin-Dependent Kinase Inhibitor p21 - metabolism

Neurons - metabolism

Neurons - drug effects

Embryonic Development - genetics

Gene Expression Regulation, Developmental - drug effects

Tumor Suppressor Protein p53 - metabolism

Morpholines - pharmacology

Genetic Techniques

Zebrafish - genetics

Artifacts

Animals

Morpholinos

Tumor Suppressor Protein p53 - chemistry

Embryonic Development - drug effects

Protein Isoforms - genetics

Details

Title: Subtitle: p53 activation by knockdown technologies
Creators: Mara E Robu - University of Minnesota, Minneapolis, Minnesota, United States of America
Jon D Larson
Aidas Nasevicius
Soraya Beiraghi
Charles Brenner
Steven A Farber
Stephen C Ekker
Resource Type: Journal article
Publication Details: PLoS genetics, Vol.3(5), pp.787-801
DOI: 10.1371/journal.pgen.0030078
PMID: 17530925
PMCID: PMC1877875
NLM abbreviation: PLoS Genet
ISSN: 1553-7390
eISSN: 1553-7404
Publisher: United States
Grant note: R01 CA075954-10 / NCI NIH HHS\r\nT32 DE007288 / NIDCR NIH HHS\r\nCA075954 / NCI NIH HHS\r\nR01 GM063904-01 / NIGMS NIH HHS\r\nR01 GM063904 / NIGMS NIH HHS\r\nGM63904 / NIGMS NIH HHS\r\nT32-DE07288-07 / NIDCR NIH HHS\r\nR01 CA075954 / NCI NIH HHS\r\nR56 GM063904 / NIGMS NIH HHS
Language: English
Date published: 05/25/2007
Academic Unit: Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9983788598702771

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