Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Nucleic Acids Res. 2011 Nov 1;39(20):e133. doi: 10.1093/nar/gkr628. Epub 2011 Aug 8.
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.
许多基因在发育过程中具有多种、有时是不同的功能。因此，为了在发育过程中进行功能分析而进行基因敲低的时间必须要有精确的时间控制，因为在早期阶段失去一个基因的功能会阻止其在发育后期的分析。RNAi 与鸡胚的可及性相结合，是在神经发育过程中进行基因功能的时间控制分析的有效方法。在这里，我们描述了新型质粒载体，其包含细胞类型特异性启动子/增强子，以驱动荧光标记物的表达，然后直接表达 miR30-RNAi 转录本以进行基因沉默。这些载体允许通过明亮的荧光直接追踪经历基因沉默的细胞。敲低水平足以重现已知具有轴突导向功能的基因受到干扰时出现的预期轨迹缺陷。在电穿孔之前混合不同的载体，可以在脊髓的不同区域同时敲低多个基因。这使得能够以快速和精确的方式检查发育过程中的复杂细胞和分子相互作用。我们描述的鸡胚内 RNAi 技术的改进不仅将显著增强鸡中的功能基因分析，还可以适应发育研究中的其他生物体。
