Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
Methods. 2012 Jan;56(1):50-4. doi: 10.1016/j.ymeth.2011.10.001. Epub 2011 Oct 13.
Myogenesis has proven to be a powerful paradigm for understanding cell fate specification and differentiation in many model organisms. This includes the nematode Caenorhabditis elegans for which the genetic, cellular, and molecular tools have allowed an in-depth understanding of muscle development. One tool not yet available in C. elegans is a robust, pure and prolific cell culture system to study myogenesis. As an alternative, this chapter describes a method by which the cell fates of early, uncommitted blastomeres in the embryo are converted to a myogenic lineage. This technique permits the nearly synchronous induction of myogenesis in vivo with the potential to generate a nearly homogeneous population of cells. Coupled with the RNA isolation and cDNA amplification methods that are also described, one can now profile gene expression throughout myogenesis using any platform of choice (e.g. expression arrays, next generation sequencing). Although limited by the artificial nature of this developing mass of muscle inside the eggshell, blastomere conversion and transcriptional profiling is a very powerful tool to investigate changes in gene expression associated with myogenesis in C. elegans that is applicable to many different cell types. When coupled with next generation sequencing, the method has the potential to yield a very high-resolution map of changes in gene expression throughout myogenesis.
肌发生已被证明是一个强大的范例，可以帮助我们理解许多模式生物中的细胞命运特化和分化。这包括线虫秀丽隐杆线虫，其遗传、细胞和分子工具使我们能够深入了解肌肉发育。秀丽隐杆线虫中尚未具备的一个工具是一种强大、纯净且多产的细胞培养系统，用于研究肌发生。作为替代方法，本章描述了一种将胚胎早期未特化的卵裂球的细胞命运转化为肌发生谱系的方法。该技术可在体内近乎同步诱导肌发生，并有可能产生几乎同质的细胞群体。与本文中还描述的 RNA 分离和 cDNA 扩增方法相结合，现在可以使用任何首选的平台（例如表达谱芯片、下一代测序）来分析整个肌发生过程中的基因表达。尽管这种在蛋壳内发育的肌肉块具有一定的人为性质，但卵裂球转化和转录谱分析是一种非常强大的工具，可以研究秀丽隐杆线虫中与肌发生相关的基因表达变化，适用于许多不同的细胞类型。当与下一代测序相结合时，该方法有可能生成整个肌发生过程中基因表达变化的高分辨率图谱。
