建立稳定抑制死亡相关蛋白激酶表达的细胞系

基础医学与临床 ›› 2008, Vol. 28 ›› Issue (3): 222-226.

建立稳定抑制死亡相关蛋白激酶表达的细胞系

郭黠汪亚君张海涛

广东医学院广东医学院广东医学院

收稿日期:2007-04-09 修回日期:2005-07-02 出版日期:2008-03-25 发布日期:2008-03-25
通讯作者: 张海涛

Establishment of PC12 cell lines stably suppressing the DAPK expression

Xia GUO, Ya-jun WANG, Hai-tao ZHANG

Received:2007-04-09 Revised:2005-07-02 Online:2008-03-25 Published:2008-03-25
Contact: Hai-tao ZHANG

摘要/Abstract

摘要： 目的筛选稳定抑制DAPK表达的PC12细胞系，并观察这些细胞系的生长特性。方法设计、合成4条RNA干扰序列，连接到pDsRed1-N1-U6质粒载体上，扩增后提取质粒转染到PC12细胞，并同时转染1株空载质粒作为对照，然后通过G418筛选细胞克隆，建立稳定增殖细胞系，再通过Western blot筛选最为有效的干扰序列，最后用MTT，流式细胞术等检测转染细胞系的生长特性。结果 4条干扰序列均已成功转染并筛选出单克隆细胞系，Western blot实验结果证实，4条干扰序列中有3条对DAPK的表达有明显的抑制作用，其中以第2条干扰序列的抑制效果最为明显结论成功建立稳定增殖和抑制DAPK表达的PC12细胞系。

Abstract: Aim To establish the stable inhibition of DAPK gene expression PC12 cell lines and observate the character Methods 5 pairs of shRNA were designed by using web soft ware, then they were synthesized and inserted into the pDsRed1-N1-U6 vector. The recombinant plasmids were transfected into PC12 cell and screening the monoclone by G418 and establishing the stable PC12 cell lines. DAPK expression was detected by Western blot. MTT, FCM assay were used to assess the cell lines growth. Results The recombinant plasmids were successfully constructed and transfected into PC12 cell, Western blot showed that the RNA interference effect of the pDsRed1-N1-U6-F2 was the best among them. Conclusion It is a feasible way to establish the stable inhibition of DAPK gene expression PC12 cell lines by shRNA expression vectors.

Key words: DAPK, RNAi, shRNA expression vectors, PC12 cell

郭黠汪亚君张海涛. 建立稳定抑制死亡相关蛋白激酶表达的细胞系[J]. 基础医学与临床, 2008, 28(3): 222-226.

Xia GUO; Ya-jun WANG; Hai-tao ZHANG. Establishment of PC12 cell lines stably suppressing the DAPK expression[J]. Basic & Clinical Medicine, 2008, 28(3): 222-226.