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Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors

Research output: Contribution to journalArticle

  • Michael Mende
  • Nicolas A D Christophorou
  • Andrea Streit

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Original languageEnglish
Pages (from-to)947-62
Number of pages16
JournalMechanisms of Development
Volume125
Issue number11-12
DOIs
Publication statusPublished - 2008

Abstract

In the chick embryo, two methods are now used for studying the developmental role of genes by loss-of-function approaches: vector-based shRNA and morpholino oligonucleotides. Both have the advantage that loss-of-function can be conducted in a spatially and temporally controlled way by focal electroporation. Here, we compare these two methods. We find that the shRNA expressing vectors pRFPRNAi, even when targeting a non-expressed protein like GFP, cause morphological phenotypes, mis-regulation of non-targeted genes and activation of the p53 pathway. These effects are highly reproducible, appear to be independent of the targeting sequence and are particularly severe at primitive streak and early somite stages. By contrast, morpholinos do not cause these effects. We propose that pRFPRNAi should only be used with considerable caution and that morpholinos are a preferable approach for gene knock-down during early chick development.

    Research areas

  • Animals, Chick Embryo, DNA Primers, Electroporation, Gene Expression Regulation, Gene Knockdown Techniques, Gene Silencing, Genes, p53, Genetic Techniques, Genetic Vectors, Green Fluorescent Proteins, Luminescent Proteins, Models, Biological, Oligonucleotides, RNA Interference

ID: 13093628