[Construction and identification of Mdr-1-shRNA eukaryotic expression vector].
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.
Department of Hematology, Zhongda Hospital, Southeast University Clinical Medical College, Nanjing 210009, Jiangsu Province, China.
Pei-Pei
PP
Mao
Bao-An
BA
Chen
Jian
J
Cheng
Feng
F
Gao
Jia-Hua
JH
Ding
Chong
C
Gao
Yun-Yu
YY
Sun
Jun
J
Wang
Gang
G
Zhao
Wen
W
Bao
Hui-Hui
HH
Song
Wei
W
Zhang
Wei-Wei
WW
Wu
Guo-Hua
GH
Xia
-Wen-Lin
-L
Xu
Xue-Mei
XM
Wang
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D034741:RNA, Small Interfering