The serine-threonine protein kinase PAK4 is dispensable in zebrafish: identification of a morpholino-generated pseudophenotype.

Sheran H W Law,Thomas D Sargent,Bruce Riley

doi:10.1371/journal.pone.0100268

Abstract

TALEN-based inactivation of the zebrafish pak4 gene resulted in embryos and adult fish that appear normal and fertile. This is in contrast to our previously published studies which were based on the use of antisense morpholino oligonucleotides (MOs). We have excluded potential explanations such as gene duplication, alternate splicing, cryptic initiation of translation, and translation-independent RNA function. Our conclusion is that pak4 is dispensable in zebrafish, and that even when corroborated by robust controls, such as RNA rescue, MOs may elicit misleading pseudophenotypes that do not correspond to results obtained by genetic mutations, and should thus be used with caution.

Highlights

The optical clarity, rapid, in vitro embryonic development and relatively short generation time of zebrafish (Danio rerio) have led to the extensive use of this species for the characterization of gene function in vertebrate development
Genotype analysis of the 1month-old F1 fish revealed an average of 10% germ-line transmission of mutant alleles, with the same mutant allele present in all individuals derived from a single G0 parent
It is reasonably clear that the locus we have studied in fish is the closest homolog to the mammalian pak4 [8], but are these genes functional homologs? The genetics would seem to indicate that they are not

Summary

Introduction

The optical clarity, rapid, in vitro embryonic development and relatively short generation time of zebrafish (Danio rerio) have led to the extensive use of this species for the characterization of gene function in vertebrate development. The majority of gene function studies in zebrafish have been based on the use of antisense oligonucleotides with degradation-resistant morpholino phosphorodiamidate backbones (MOs). These have been shown to interact with complementary RNA targets, resulting in inhibition of translation, RNA splicing, and other steps in posttranscriptional gene regulation (e.g Lim et al, 2012 [3]). In addition to cell death, MO-induced p53 can elicit patterned gene expression via activation of proapoptotic factors, that can be mistaken for developmental regulation [7]. If the MO-induced phenotype can be reverted to the control pattern, the knockdown has been deemed to result from the loss of target gene expression. We used MOs, in combination with both of these types of controls, to analyze the developmental function of the serine/ threonine protein kinase PAK4 in zebrafish [8]

Methods

Results

Conclusion