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.

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye

“…Fig. S1C,D; Robu et al, 2007), suggesting that the gene expression changes are directly caused by the mutation.…”

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish

“…Acridine orange staining showed similar patterns of apoptosis (data not shown). It is known that offtarget effects of MOs could be mediated through tumor suppressor p53-induced apoptosis (Robu et al, 2007). As the p53 protein was shown to be up-regulated in the microarray analysis of gr ATG1 MO embryos at 5 and 10 hpf (see below), we examined the occurrence of possible off-target effects by co-injecting an anti-p53 MO with gr ATG1 MO or gr ATG2 MO.…”

“…An antisense splice variant MO was designed on the sequence at the exon 3-intron 3-junction, MO4-nr3c1 (gr splic MO): 5 0 -GCC AGA GAT ATA TGG AAT ACC TTC A-3 0 (the exon complementary sequence is underlined). Finally, we used an MO against the ATG translation initiation site of p53 mRNA (MOp53): 5 0 -GCG CCA TTG CTT TGC AAG AAT TG-3 0 (named MO4-tp53 by Robu et al, 2007).…”

Evolutionarily conserved function of Gbx2 in anterior hindbrain development

“…It has been shown that non-specific cell death caused by morpholinos can be due to an upregulation of p53, which modulates a variety of pro-apoptotic genes (Miyashita et al, 1994;Roperch et al, 1998;Robu et al, 2007). To test whether the cell death in r2, r3, and r5 is specific to gbx2 knockdown, we co-injected embryos with p53-MO to minimize non-specific apoptosis.…”

“…A dose of 8 ng was used. For p53 morpholino, we used a concentration of 3 ng/nl as previously described (Robu et al, 2007). We estimated the dose per embryo based upon the concentration of the MO solution, and the diameter (volume) of the injection bolus in the yolk cell.…”