随着基因组测序项目的完成,发育生物学家面临的一个新挑战是将功能分配给成千上万个已鉴定的基因。外源mRNA的表达是一个强大的,可用于研究海胆发育过程中的基因功能的通用和快速技术。本章介绍了该技术如何用于分析棘皮动物胚胎中的基因功能,如何将其与细胞移植结合以进行镶嵌分析,以及如何将其应用于识别转录因子和信号通路的下游靶基因。我们描述了使用mRNA过表达来分析基因功能的具体例子,提到了该技术的益处和目前的局限性,并强调了使用不同对照来评估所观察到的效应的特异性的重要性。最后,本章详细介绍了不同的步骤,用于体外产生mRNA和表型分析的载体和方案。
With the completion of the genome sequencing projects, a new challenge for developmental biologists is to assign a function to the thousands of genes identified. Expression of exogenous mRNAs is a powerful, versatile and rapid technique that can be used to
study gene function during development of the sea urchin. This chapter describes how this technique can be used to analyze gene function in echinoderm embryos, how it can be combined with cell transplantation to perform mosaic analysis and how it can be applied to identify downstream targets genes of transcription factors and signaling pathways. We describe specific examples of the use of overexpression of mRNA to analyze gene function, mention the benefits and current limitations of the technique and emphasize the importance of using different controls to assess the specificity of the effects observed. Finally, this chapter details the different steps, vectors and protocols for in vitro production of mRNA and phenotypic analysis.