One of the RNA silencing products available from Ambion is a kit for the synthesis of siRNA, the Silencer™ siRNA Construction Kit. While this kit is very cost effective when RNAs of several different sequences need to be made, it can be advantageous to turn to chemical synthesis once a specific siRNA sequence is needed in bulk. Ambion also offers a Custom siRNA Oligonucleotide Synthesis Service. siRNA oligonucleotide pairs are provided after optimized deprotection and reverse phase desalting, and for a limited time, free PAGE purification. Here Milhavet and Mattson, researchers at the NIH, use PAGE purified siRNAs from Ambion to study gene function during mouse neural stem cell differentiation.

Ollivier Milhavet and Mark P. Mattson
Laboratory of Neurosciences,
National Institute on Aging, Gerontology Research Center,
National Institutes of Health, Baltimore, MD 21224

RNA interference (RNAi) is the phenomenon by which double-stranded RNA (dsRNA) specifically suppresses the expression of a gene with homologous sequence at the post-transcriptional level. In C. elegans, D. melanogaster and mammalian cell cultures this effect has been shown to be mediated by 21­23 nucleotide 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 37°C 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 ß-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 neural stem cells, which grow as neurospheres in the culture medium, a 50% reduction in protein synthesis of the target gene was observed. The lower efficiency of gene silencing in these primary culture cells can be attributed to inefficient transfection observed with cells grown in suspension and poor transport of the siRNAs to the core of the sphere. Cells transfected with Oligofectamine alone or single-stranded RNA as controls did not show altered levels of the targeted protein. Moreover, nonspecific RNAi effects, such as activation of the PKR pathway, were not observed. In some cases, when growth factors were removed from the culture medium, differentiation of the primary neural stem cells, which normally give rise to neuronal and glial cells, was altered in the siRNA treated cells. siRNA-mediated gene silencing is thus proving to be a powerful tool for the study of differentiation of mouse neuronal primary cells.

References

1. Zamore PD, Tuschl T, Sharp Pa, Bartel DP. (2000) RNAi:
double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 101(1): 25­33.

2. Elbashir SM, Lendeckel W, and Tusch T (2001) RNA interference is mediated by 21- and 22- nucleotide RNAs. Genes Dev. 15(2): 188­200.

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