RNA Interference Conferred by Retrotransposable Alu Sequence
Date Issued
2005
Date
2005
Author(s)
Soh, Gim-Hooi
DOI
zh-TW
Abstract
RNA interference, a phenomenom where dsRNA specifically blocks the expression of its homologous gene, is discovered by Fire and Mell in 1998. It is clear that RNAi occurs in all eukaryotic cells and plays a role in viral defense and transposon silencing mechanisms. RNAi becomes an important method for analyzing gene functions and development of therapeutic gene silencing. Alu sequence is primate-specific member of the SINE（short interspersed element） retrotransposon family，and comprises about 15﹪ of human genome. Full –length Alu sequence is about 250∼300bp long and is commonly found in intron，3’ untranslated regions of genes and intergenic regions. Alu sequence was ancestrally derived from the 7SL RNA gene. Mutations accumulated in the Alu sequence caused it classified of several distinct subfamily. We used H1 promoter amplified from human genomic DNA and synthetic TTTTT as terminator to construct our siRNA expression vector. It can reduce GFP expression after constructed as siGFP expression vector. This means that the constructed siRNA expression vector is functional. After searching the Alu sequence containing genes from the database, we chose gene HSD17B12、OACT1、LOC134218、LOC146909 and SMG1 to research. After amplifying the trancated Alu sequence from the gene HSD17B12, we insert it into the vector as a inverted repeat to construct siAlu expression vector,named pH1-siAlu95. After transfecting into cells, it resulted in reducing expression of GFP gene containing Alu sequence in 3’ end, showed that Alu sequence can serve as RNAi target.
After examination, HEK293, H1299, SC-M1 and K562 cells have expressed the five Alu containing genes and we chose HEK293 for continuous research. Our results showed that the expression of gene HSD17B12 and OACT1 are repressed after transfection of pH1-siAlu. It demonstrated that Alu sequence can serve as RNAi target. To use Alu sequence for RNAi target can simultaneously downregulate several genes by redused the expression level of mRNA and protein﹒
Subjects
反轉錄跳躍子
Alu序列
核醣核酸干擾現象
retrotransposon
Alu sequence
RNA interference
siRNA
Type
other