Transgenesis by microparticle bombardment for live imaging of fluorescent proteins in Pristionchus pacificus germline and early embryos
Transgenesis by microparticle bombardment for live imaging of fluorescent proteins in Pristionchus pacificus germline and early embryos
复制标题
DOI：
10.1007/s00427-018-0605-z
复制
发表时间：
2018-01-01
影响因子：
2.4
通讯作者：
Sugimoto, Asako
中科院分区：
文献类型：
--
作者：
Namai, Satoshi;Sugimoto, Asako
Pristionchus pacphaus是一种自由生活的线虫，是进化、发育和生态生物学的模式生物。虽然已经建立了一种通过显微注射形成复杂阵列的转基因技术，但该阵列在生殖系和早期胚胎中的转基因表达往往是沉默的。在这里，我们建立了一种利用潮霉素B选择的微粒子轰击将转基因整合到太平洋假单胞菌基因组中的方法。此外，我们分离到了一个突变体，它在生殖系和早期胚胎中表现出显著的低自发荧光，便于将转基因衍生的荧光蛋白用于活体成像。使用这些工具构建的转基因株系成功地在生殖系和早期胚胎中表达了GFP标记的蛋白，并能够对染色体、微管和中心体进行实时成像。
Pristionchus pacificus is a free-living nematode used as a model organism for evolutionary developmental and ecological biology. Although a transgenic technique to form complex arrays by microinjection has been established in P. pacificus, transgene expression from the array in the germline and early embryos tends to be silenced. Here, we established a method to integrate transgenes into the genome of P. pacificus using microparticle bombardment with hygromycin B selection. Additionally, we isolated a mutant exhibiting significantly lower autofluorescence in the germline and early embryos, facilitating visualization of transgene-derived fluorescent proteins for live imaging. Transgenic lines constructed using these tools successfully expressed GFP-tagged proteins in the germline and early embryos and enabled live imaging of chromosomes, microtubules, and centrosomes.