Screening of effective shRNA targeting TNF-alpha and constructing of recombinant plasmid / 中国实验血液学杂志
Journal of Experimental Hematology
;
(6): 180-183, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-302171
ABSTRACT
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Recombination, Genetic
/
RNA, Messenger
/
Transfection
/
Genetic Therapy
/
Gene Expression
/
Cells, Cultured
/
Tumor Necrosis Factor-alpha
/
Cell Biology
/
RNA, Small Interfering
Type of study:
Diagnostic study
/
Prognostic study
/
Screening study
Limits:
Animals
Language:
Chinese
Journal:
Journal of Experimental Hematology
Year:
2009
Type:
Article
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