[Construction of pEGFP-C1/U6-mediated plasmid expressing MDR1 shRNA].
To construct a plasmid expressing MDR1 short hairpin RNA (shRNA) mediated by pEGFP-C1/U6 vector, two coding sequences of 19 nucleotides were selected from MDR. Two pairs of oligonucleotides were designed for these two fragments. After annealing the formed double-stranded DNAs were ligated with plasmid pEGFP-C1/U6 (pEGFP-C1 vector with U6 promoter). The plasmids producing MDR1 shRNA were constructed from the inverted motif containing 9 spacers and four Ts. The results showed that the constructed plasmids were named pEGFP-C1/U6/A and pEGFP-C1/U6/B, and the constructs were identified by restriction and sequence analysis, no any base mutation was observed. It is concluded that plasmids of pEGFP-C1/U6/A and pEGFP-C1/U6/B expressing MDR1 shRNA were successfully constructed, providing a highly effective method for reversing the multidrug resistance in clinic.
Xiao
Xi-Bin
XB
Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, China.
Xie
Zhao-Xia
ZX
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D049452:Green Fluorescent Proteins