Publisher: Spandidos Publications
E-ISSN: 1792-1015|10|6|2283-2288
ISSN: 1792-0981
Source: Experimental and Therapeutic Medicine, Vol.10, Iss.6, 2015-01, pp. : 2283-2288
Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.
Abstract
The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and cotransfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNFα mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNFα protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)1β and IL6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2x106 TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNFα gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for the in vivo study of RNAi gene therapy targeting TNF-α.
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