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[Construction of eukaryotic expression vector of siRNA specific to bcr/abl fusion gene]
Ya-ming Xi1, Ting Liu, Wen-tong Meng
1Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Objective:
To construct eukaryotic expression vector of siRNA specific to bcr/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells.
Methods:
siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs)of siRNA for bcr/abl fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, identified by the restriction map and the sequence analysis, and transfected into K562 cells by Lipofectamine. Expression of bcr/abl mRNA was assayed by RT-PCR; expression of P210 protein was detected by immunohistochemistry.
Results:
The pTER117 and pTER363 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells, the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein(P210) in K562 cells.
Conclusion:
The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed, and it effectively inhibits the expression of bcr/abl in K562 cells.
