P53 activation by knockdown technologies

Farber, Stephen C. 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 pdependent 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.

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 pdependent 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 pdependent cell death pathway. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. N2 - Morpholino phosphorodiamidate antisense oligonucleotides MOs and short interfering RNAs siRNAs are commonly used platforms to study gene function by sequence-specific knockdown.

Pediatric Dentistry Chancellor's Office Rochester. Overview Fingerprint. 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.