Department for Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, A-1030, Vienna, Austria.
Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, 69120, Heidelberg, Germany.
BMC Biotechnol. 2018 Jan 9;18(1):1. doi: 10.1186/s12896-017-0411-0.
The application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene.
A single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes.
Our results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation.
反义分子（如吗啉代寡核苷酸）的应用是体内基因失活的有效方法。我们最近引入了磷酸酯修饰的肽核酸（PNA），用于进行斑马鱼胚胎体内的功能丧失实验。在这里，我们测试了 PNA 骨架的新型修饰，以敲低斑马鱼 tcf3 基因。
在斑马鱼基因组中存在单个 tcf3 基因，其失活强烈影响胚胎的眼睛发育，导致眼睛大小缩小和严重情况下的无眼症。Tcf3 的功能强烈依赖于共抑制因子的相互作用。我们发现与 Groucho/Tle 蛋白的相互作用对眼睛发育最重要。使用显性负性方法联合失活所有 Groucho/Tle 基因也会导致眼睛表型，干扰三个单独的 tle 基因也是如此。
我们的结果表明，侧链修饰的 PNA 与体内的 morpholino 寡核苷酸的敲低效率非常接近。单个斑马鱼 tcf3 基因结合了两个斑马鱼同源物 hdl 和 tcf3b 的功能。与 Groucho/Tle 共抑制蛋白结合，Tcf3 在前端发育中起作用，对眼睛形成至关重要。
