siRNA-Mediated Gene Silencing in Neural Stem Cells

Take small interfering RNA, or siRNA, a type of RNA involved in gene silencing.

Add spherical lipid vesicles called liposomes.

The liposome encapsulates the siRNA, protecting it from degradation.

Add siRNA-liposome complexes to a culture well containing neural stem cells. Keep one well as a control, and incubate.

The liposome fuses with the cell membrane, releasing the siRNA.

The siRNA binds to the RNA-induced silencing complex, or RISC, where one strand is removed.

The remaining strand targets the mRNA responsible for cell differentiation, leading to its degradation.

Add a suitable differentiation medium to the wells.

In the control well, the growth factors and other nutrients in the medium promote the differentiation of neural stem cells into osteoblasts.

Using suitable staining techniques, stain osteoblast-specific markers on the cells and stain the nucleus with a blue fluorescent dye.  

The absence of osteoblast-specific markers in the gene-silenced cells indicates their inability to differentiate.

To perform siRNA knockdown in O9-1 cells recover and seed the cells. Dilute liposomes in an appropriate volume of serum-free media. Then, dilute siRNA in serum-free media according to the text protocol. After this, add the diluted siRNA to the diluted liposomes according to the manufacturer's instructions. Mix the solution by pipetting and incubate for 5 minutes at room temperature.

Next, add an appropriate volume of siRNA-lipid complex to the cells. Then, incubate the cells for 24 hours in a standard cell culture incubator. O9-1 cells can differentiate into different cell types under specific differentiation culture conditions. To differentiate O9-1 cells into osteoblasts, prepare osteogenic differentiation media according to the text protocol. Finally, detect the osteoblast marker osteocalcin in differentiated osteoblasts by immunostaining.