Poster: Posttranscriptional Regulation

Abs # 1078: Studies on double stranded RNA mediated Gene silencing.

Presenter:	Unnamalai, Narayanan , narayanan2@yahoo.com
Authors	Unnamalai, Narayanan  (A)   Woo Sung, Lee  (A)
Affiliations:	(A): Unnamalai,N, Hyun Hee, Ji Hyun, Woo Sung Lee, SungKyunKwan University

Studies on double stranded RNA mediated gene silencing Abstract Double stranded RNA (dsRNA) was used to silence the reporter gene, GUS in tobacco suspension cells transformed with pBI121 having GUS gene under 35S promoter. Gene silencing studies in plants were carried out by transforming plants with a vector carrying sense or antisense or sense and antisense orientation of the gene to be silenced. In this study we used a direct nucleic acid transformation system to deliver dsRNA into suspension cells. The northern blot analysis showed that 20µg of dsRNA was effective in silencing the gene in 24h. and there was no silencing in the cells treated with dsRNA alone indicating that the peptide efficiently delivered the dsRNA into the cells. we further investigated the formation of smaller RNA by northern blot analysis. We could not detect the presence of small RNA about 21~23 nucleotide which was reported to initiate the gene silencing in plants. The peptide has the property of nuclear localization. Hence we checked the localization of dsRNA in the treated cells. We labeled the dsRNA with FITC and studied the localization of the dsRNA in the treated cells by fluorescence microscopy. The localization of the dsRNA was seen around the nuclear membrane and in the cytoplasm. It indicates that mRNA is degraded in the cytoplasm. Also the day until which the silencing effect was exhibited in dsRNA treated cells was determined. There was no mRNA to GUS detected in the treated cells until 21 days by RT-PCR. The silencing effect was there for three weeks.