siRNA mediated gene silencing studies were carried out to assess the mechanisms of mouse neural stem cell differentiation. Silencing of Gadd153, a pro-apoptotic transcription factor, was first assessed in a mouse neuroprecursor cell line, C17.2. Chemically synthesized, PAGE purified siRNAs (Ambion) were transfected into cells grown to 4070% confluency in complete medium without antibiotics. Individual siRNAs (2080 nM) were mixed with Oligofectamine and Opti-MEM (Invitrogen) and incubated at room temperature for 20 min. This mixture was added to a minimal volume of serum-free medium and transferred to C17.2 cells. The cells were incubated for 4 hr at 37C after which medium containing serum was added. Subsequent to the addition of serum, the cells were incubated for 15 days and harvested for further analysis. Western analyses revealed 7585% silencing of the Gadd153 gene (Figure 1).
Figure 1. Gene Silencing in a Neuroprecursor Cell Line Using siRNA. siRNAs were designed to target Gadd153, a pro-apoptotic transcription factor, and were transfected into C17.2 cells using Oligofectamine and Opti-MEM (Invitrogen). Silencing efficiency was analyzed by Western blot. Control cells were treated with Oligofectamine alone, and -actin was used as a loading control. Quantitative analysis showed a knockdown efficiency of approximately 7585%.
When siRNAs targeting other proteins (to be published) were also tested on embryonic mouse primary ne