Construction siRNA expressing vector of Skp2 and the inhibitory effects on the expression of Skp2 in laryngeal carcinoma cell line Hep2 cell.
- Author:
Xiuying LU
1
;
Xiaoming LI
Author Information
1. Department of Otorhinolaryngology-Head and Neck Surgery, Bethune International Peace Hospital of PLA, Shijiazhuang, 050082, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Cell Line, Tumor;
Genetic Vectors;
Humans;
Laryngeal Neoplasms;
genetics;
Plasmids;
RNA Interference;
RNA, Messenger;
genetics;
RNA, Small Interfering;
genetics;
S-Phase Kinase-Associated Proteins;
genetics;
Transfection
- From:
Journal of Clinical Otorhinolaryngology Head and Neck Surgery
2008;22(18):846-849
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct the siRNA expression vector of Skp2 and inhibit the expression of Skp2 through RNA interference in laryngeal carcinoma cell line Hep2 cell.
METHOD:According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGPU6/Neo siRNA expression vector, and the recombinant plasmid was transformed into strain DH5a. The plasmid identified by restriction enzyme was used for sequencing. After being identified by sequencing, the recombinant plasmids pGPU6Skp2 were transfected into Hep2 cells. Skp2 expression in the transfected cells was assayed by flow cytometry.
RESULT:DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 expression in the transfected cells was down-regulated significantly by pGPU6Skp2 at the protein level.
CONCLUSION:The siRNA expression vector of Skp2 was successfully constructed and could inhibit Skp2 expression in Hep2 cells. This result will facilitate further studies of Skp2 in gene therapy for tumors.
