[Screening of effective shRNA targeting TNF-alpha and constructing of recombinant plasmid].
The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha (TNF-alpha), to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for searching gene therapy of TNF-alpha related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2 x 10(7) cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37 degrees C in a fully humidified atmosphere with 5% CO(2). Cells were stimulated with lipopolysaccharide (LPS) and the concentration of TNF-alpha in the supernatant at different time points was determined by enzyme-linked immunosorbent assay (ELISA). The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the cells were stimulated with LPS for 24 hours. The concentration of TNF-alpha in the supernatant and the expression of TNF-alpha mRNA were determined by ELISA and reverse transcription polymerase chain reaction (RT-PCR) respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-alpha in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-alpha by 59.46% and the expression of TNF-alpha mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-alpha was successfully screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-alpha related disease.
Song
Xiao-Yu
XY
Department of Pathophysiology, China Medical University, Shenyang 110001, China.
Zheng
Ning-Ning
NN
Sun
Lu-Ning
LN
Zhang
Hai-Peng
HP
D000818:Animals; D002478:Cells, Cultured; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D008264:Macrophages / Q000166:cytology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha