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Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development

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  • Nicole H Wilson
  • Esther T Stoeckli

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    Rights statement: This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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http://nar.oxfordjournals.org/content/39/20/e133.long
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203593/
Original languageEnglish
Pages (from-to)e133
JournalNucleic Acids Research
Volume39
Issue number20
DOIs
Publication statusPublished - 1 Nov 2011

Abstract

Many genes have several, sometimes divergent functions during development. Therefore, timing of gene knockdown for functional analysis during development has to be done with precise temporal control, as loss of a gene's function at early stages prevents its analysis later in development. RNAi, in combination with the accessibility of chicken embryos, is an effective approach for temporally controlled analysis of gene function during neural development. Here, we describe novel plasmid vectors that contain cell type-specific promoters/enhancers to drive the expression of a fluorescent marker, followed directly by a miR30-RNAi transcript for gene silencing. These vectors allow for direct tracing of cells experiencing gene silencing by the bright fluorescence. The level of knockdown is sufficient to reproduce the expected pathfinding defects upon perturbation of genes with known axon guidance functions. Mixing different vectors prior to electroporation enables the simultaneous knockdown of multiple genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a fast and precise manner. The advancements of the in ovo RNAi technique that we describe will not only markedly enhance functional gene analysis in the chicken, but also could be adapted to other organisms in developmental studies.

    Research areas

  • Animals, Chick Embryo, Gene Knockdown Techniques, Genetic Vectors, Luminescent Proteins, MicroRNAs, Neural Tube, Neurons, Promoter Regions, Genetic, RNA Interference, RNA Polymerase II, Spinal Cord

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