Abstract

RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for “transient transgenic” knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.

Highlights

  • The production of targeted mutations in mice remains the gold standard for the analysis of the loss-of-function studies of specific genes in mammals

  • We initiated transient transgenic RNA interference (RNAi) experiments in mice to examine the effect of reduced Prdm16 expression in E16.5 mouse embryos, by which time wild type palate shelves have elevated and fused [23]

  • We chose to pursue transient transgenic RNAi knockdown during mouse embryogenesis as a means to rapidly validate loss of function gene mutations, which we have identified as part of an ENU mutagenesis screen for late embryonic phenotypic anomalies [15]

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Summary

Introduction

The production of targeted mutations in mice remains the gold standard for the analysis of the loss-of-function studies of specific genes in mammals. Even with the emergence of largescale knockout mouse resources, such as those of the International Knockout Mouse Consortium (http://www.knockoutmouse.org/), generation of such mutants using embryonic stem (ES) cells may still require substantial time and resources. This approach is difficult to pursue for high throughput applications. Linkage and association studies for mutations or strain-specific traits may yield a large number of positional candidate genes, which may require testing individually to assess causality. An efficient methodology to rapidly screen genes in vivo would enhance the functional analysis of outputs from high throughput screening

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