Fu Xueyao, Kiyama Takae, Li Renzhong, Russell Mark, Klein William H, Mu Xiuqian
Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA.
Dev Dyn. 2009 Sep;238(9):2309-17. doi: 10.1002/dvdy.21974.
Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5(HA) and Pou4f2(HA) alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.
尽管蛋白质的免疫检测在视网膜发育研究中被广泛应用，但研究常常受到阻碍，因为针对关键蛋白质的抗体无法产生或难以获得。在此，我们通过构建视网膜神经节细胞（RGC）发育所需的两个基因Math5和Pou4f2的基因工程等位基因，克服了这些限制。将编码来自血凝素（HA）的肽表位的序列与Math5或Pou4f2框内添加，以产生Math5(HA)和Pou4f2(HA)等位基因。我们证明，标记的等位基因重现了这两个基因的野生型表达模式，并且标签不干扰同源蛋白的功能。此外，通过共染色，我们发现Math5和Pou4f2在新生RGC中短暂共表达，明确表明Pou4f2在RGC形成中直接位于Math5下游。表位标记的等位基因为分析RGC发育潜在的基因调控网络提供了新的有用工具。
