Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-60215
Wilson, N H; Stoeckli, E T (2011). Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development. Nucleic Acids Research, 39(20):e133.
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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.
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|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||07 Faculty of Science > Institute of Molecular Life Sciences|
|DDC:||570 Life sciences; biology|
|Uncontrolled Keywords:||artificial miRNA, electroporation, in vivo gene silencing, embryonic development, axon guidance|
|Date:||1 November 2011|
|Deposited On:||05 Mar 2012 12:12|
|Last Modified:||19 Jan 2014 09:43|
|Publisher:||Oxford University Press|
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