Sivakamasundari V, Kraus Petra, Jie Song, Lufkin Thomas
Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore.
Genesis. 2013 Jun;51(6):420-9. doi: 10.1002/dvg.22379. Epub 2013 Feb 25.
The Paired box gene 1 (Pax1) transcription factor plays essential roles in the development of axial skeleton, scapula, pelvic girdle, and thymus. Delineating its pleiotropic and molecular roles in the various tissues requires the ability to track and isolate the Pax1-expressing cells for downstream high-throughput experiments such as microarray and RNA-sequencing. With these applications in mind, we have generated two new mouse lines-a Pax1 wildtype (WT) mouse line that co-expresses enhanced green fluorescent protein (EGFP) with functional Pax1, and a Pax1 knockout mouse line which expresses EGFP under the control of Pax1 promoter, using the internal ribosome entry site (IRES) and 2A-peptide multi-cistron concatenating strategies. These mouse lines facilitate the isolation and enrichment of Pax1-specific cells from Pax1-positive and Pax1-null embryos using fluorescence activated cell sorting (FACS). They can be also be used in parallel to investigate the stage- and tissue-specific molecular functions of Pax1.
配对盒基因1(Pax1)转录因子在轴骨骼、肩胛骨、骨盆带和胸腺的发育中起着至关重要的作用。要阐明其在各种组织中的多效性和分子作用,需要能够追踪和分离表达Pax1的细胞,以用于下游的高通量实验,如微阵列和RNA测序。考虑到这些应用,我们利用内部核糖体进入位点(IRES)和2A肽多顺反子串联策略,生成了两种新的小鼠品系——一种与功能性Pax1共表达增强型绿色荧光蛋白(EGFP)的Pax1野生型(WT)小鼠品系,以及一种在Pax1启动子控制下表达EGFP的Pax1基因敲除小鼠品系。这些小鼠品系有助于使用荧光激活细胞分选(FACS)从Pax1阳性和Pax1缺失的胚胎中分离和富集Pax1特异性细胞。它们还可以并行用于研究Pax1的阶段和组织特异性分子功能。