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Your Data: Silencing of a Pro-apoptotic Transcription Factor Using Synthetic siRNA

cleotide RNA fragments, termed short interfering RNAs (siRNAs), which bind and tag the complementary portion of the target mRNA for nuclease digestion (1,2).

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
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