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表达的细胞系。
Objective To screen a cell line which stably suppresses DAPK expression and to observe the growth characters. Methods Four pairs of shRNA were designed, synthesized and inserted into the pDsRed1-N1-U6 vector. The recombinant plasmids were purified and transfected into PC12 cell. Meanwhile, a pDsRed1-N1-U6 vector was transfected as control. The cell clones were screened by G418, and the stable PC12 cell line was established. DAPK expression was detected by Western blot. MTT method and Flow Cytometry（FCM） assay were used to assess the growth characters of the cell line. Results The shRNAs were transfected into PC12 cell and the cell clones were successfully screened out. Of the four recombinant plasmids, the F2 was the best interfering shRNA. Beyond our expectation, the F1 recombinant plasmid had an enhanced effect on DAPK expression. Conclusion A stable PC12 cell line with stable inhibition of DAPK expression by was established using siRNA expression vectors.
出处
《基础医学与临床》
CSCD
北大核心
2008年第3期222-226,共5页
Basic and Clinical Medicine
基金
广东省卫生厅科研课题(WJ0602)
