Construction of recombinant retroviral vector of short interfering RNAs specific for macrophage migration inhibitory factor (MIF) and establishment of stable HeLa cell line with a persistent knockdown of MIF
- VernacularTitle:siRNA靶向MIF重组逆转录病毒载体构建及稳定表达细胞株的筛选
- Author:
Bo DAI
;
Dingzhang XIAO
;
Xiyong YU
- Publication Type:Journal Article
- Keywords:
Macrophage migrotion-inhibitory factors;
RNA interference;
Retroviral vector
- From:
Chinese Journal of Pathophysiology
2000;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To construct recombinant retroviral vector of short interfering RNAs (siRNA) specific for macrophage migration inhibitory factor (MIF) and to establish the stable knockdown of MIF cell line of mammalian cells by transfecting the recombinant retroviral vectors. METHODS: We synthesized oligo-nucleotides for MIF in vitro, and cloned them into retroviral vector pSuper.retro. Subsequently the plasmids were sequenced and digested to identify the construction of the recombinant retroviral vectors. The vectors RNAi were transfected into packing cell line PHOENIX, which was selected by puromycin later. HeLa cell line was infected by the virus supernatant of stable PHOENIX cell lines, and the stable HeLa cell line showed significantly to silence MIF was established by selecting with puromycin. We also compare the characters of HeLa-pSuper-mock to HeLa-pSuper-MIF cells by using migration assay, adhesion assay, soft agar assay and FACS analysis of the cell-cycle progression. RESULTS: The recombinant retroviral vectors were constructed successfully. The HeLa cell line infected by the supernatant containing the retrovirus of package PHOENIX cells was persistent knockdown of MIF confirmed by Western blotting. Knockdown of MIF in HeLa cells inhibited the migration and adhesion, and decreased the clone formation. FACS analysis revealed that knockdown of MIF arrested HeLa cells in G0/G1 phase. CONCLUSION: We establish the stable HeLa cell line with a persistent knockdown of MIF. Our current studies reveal that MIF is necessary for HeLa cell migration and anchorage-independent growth.
