Construction and identification of an RNA interference lentiviral vector targeting the mouse TNF-α gene

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 co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-α 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 TNF-α 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 IL-6 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 2×10⁶ TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-α 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.

Keywords: RNA interference; lentiviral vector; macrophages; tumor necrosis factor-α.

Figure 1.

Relative mRNA expression levels of TNF-α, IL-β and IL-6 in RAW264.7 cells of different groups. Data are expressed as the mean ± standard error of the mean. *P<0.05 vs. negative control. TNF-α, tumor necrosis factor-α; IL, interleukin; siRNA, small interfering RNA.

Figure 2.

TNF-α protein expression levels of different RAW264.7 groups. Data are expressed as the mean ± standard error of the mean.**P<0.01 vs. negative control. TNF-α, tumor necrosis factor-α; siRNA, small interfering RNA.

Figure 3.

Electrophoresis map of the polymerase chain reaction products. Lane 1, non-constructed vector negative control; lane 2, Marker; lane 4, negative clone; lanes 3 and 5–7, positive clones.

Figure 4.

Recombinant plasmid DNA sequencing graph (part). The underlined region indicates the detected insertion sequence.

Figure 5.

Green fluorescent protein expression of 293T cells transfected with lentiviral plasmids. (A) Light microscopy; (B) fluorescence microscopy (multiplicity of infection=0.5; scale bar=200 µm).