Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Abstract

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

“…( B ) Primers sequences for qRT-PCR, ChIP and ChIP-chip validation experiments. ( C ) Sequences of synthesized siRNAs, and clone ID numbers for shRNAs obtained from Open Biosystems (the luciferase siRNA sequence was previously reported (Elbashir et al, 2001)).…”

Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2

“…Oligonucleotides used for constructions of shRNAs against cyclins B1 and B2, and for the cyclin B2/shRNA-resistant vector Double-stranded shRNA corresponding to the cDNA sequences of human cyclin B1 (5 0 -GAACAGCUCUUG GGGACAU-3 0 , nucleotides 220-238) and cyclin B2 (5 0 -UGAAACCUACUGCUUCUGU-3 0 , nucleotides 284-302) were designed as recommended (Elbashir et al, 2001). The cyclin B1 sequence was homologous to a validated Xenopus cyclin B1 siRNA (Zhou et al, 2002).…”

Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53

“…The design and application of siRNA specific for survivin mRNA were performed as described by Elbashir et al 18 The sequence of the double-stranded survivin-specific siRNA corresponded to a 21-bp sequence that began 42 bp downstream of the start codon of the survivin gene (Table 2). We also treated cells with nonsense siRNA in which the antisense and the sense sequences of the survivin-specific siRNA were exchanged (Table 2), as suggested by Tuschl et al 33 To ensure that the survivin-specific siRNA and the nonsense siRNA sequences would not interact with other transcripts, we used the sequences of these two types of RNA to perform a BLAST search against the human genome database (http://www.ncbi.nlm.nih.gov/).…”

“…[8][9][10][11][12][13][14][15][16] In addition to the use of antisense oligonucleotides and ribozymes, the application of small interfering RNA (siRNA) is an effective way to silence gene transcription. 17,18 Transcription silencing is thought to be a defense strategy that preserves genomes through evolution from attack by double-stranded RNA viruses and transposons. 19,20 Genetic and biochemical studies have confirmed that the mechanisms of gene silencing by siRNAs, cosuppression, and viruses share similarities.…”