Construction and identification of Mdr-1-shRNA eukaryotic expression vector.
- Author:
Pei-Pei MAO
1
;
Bao-An CHEN
;
Jian CHENG
;
Feng GAO
;
Jia-Hua DING
;
Chong GAO
;
Yun-Yu SUN
;
Jun WANG
;
Gang ZHAO
;
Wen BAO
;
Hui-Hui SONG
;
Wei ZHANG
;
Wei-Wei WU
;
Guo-Hua XIA
;
-Wen-Lin XU
;
Xue-Mei WANG
Author Information
1. Department of Hematology, Zhongda Hospital, Southeast University Clinical Medical College, Nanjing 210009, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
ATP Binding Cassette Transporter, Sub-Family B;
ATP-Binding Cassette, Sub-Family B, Member 1;
genetics;
Drug Resistance, Multiple;
genetics;
Drug Resistance, Neoplasm;
genetics;
Gene Expression;
Genetic Vectors;
Humans;
K562 Cells;
Plasmids;
RNA, Small Interfering;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2010;18(1):127-131
- CountryChina
- Language:Chinese
-
Abstract:
This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.
