可诱导的稳定表达Spata3细胞系的建立及特征(英文)

Establishment and Characterization of Inducible Cell Line with Stable Expression of Spata3

spata3是一个在睾丸中特异性表达的基因,可能与精子发生或生精细胞凋亡相关。为了进一步研究Spata3的功能,将spata3克隆入经修饰的pcDNA5/FRT/TO表达载体,应用Flp-InTMT-RExTM-293细胞系作为宿主细胞,成功地构建了可被四环素或Doxycline诱导的稳定表达Flp-InTMT-RExTM-sptat3的细胞系。该细胞系在spata3基因的3'端有2×FLAGtag和2×Histag,在缺乏可利用的spata3或其抗体的情况下,也能够很容易地应用商品化的FLAG抗体检测到spata3全长蛋白的表达。这种可诱导的稳定表达Flp-InTMT-RExTM-spata3的细胞系的建立,不仅有利于spata3的分析鉴定和功能研究,而且对于其他蛋白质的分离纯化和功能研究也有很好的借鉴作用。

Spata3 is a testis-specific expression gene and may play a role in spermatogenesis or spermatogenesis cell apoptosis. To further investigate the spata3 function, we successfully established the cell line which can stably express Flp-In^TMT-REx^TM-spata3 under the induction of tetractcline or doxycline by cloning the spata3 ORF into our modified pcDNA5/FRT/TO expression vector, with the Flp-In^TMT-REx^TM-293 cell line as a host line. This cell line has 2×FLAG tag and 2×His tag in the 3＇- prime of spata3 and the full-length protein of spata3 can therefore be easily detected by Western blotting with FLAG tag and/or His tag antibody. This procedure is not only very useful for spata3 functional study, but also a useful reference for other protein complex（s） purification and function identification by using highly specific anti-FLAG antibody.