toolsofmoleculargenetics-final20141217重点分析.pptVIP

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * The application of RNAi to the medical genetics RNA interference (RNAi) In 1990 . In the petunia “cosuppression” In 1994. In the fungi “quelling” In 1995. In C.elegans sense RNA can block the expression of par-1 gene In 1998. Fire and Mello suggested new mechanism for the phenomenon of gene silencing-RNAi The phenomenon of RNA inference - universality What is RNAi ? RNA interference (RNAi) – RNAi leading to the selective degradation of target mRNA and knockdown of gene function is induced by introducing into cells a short dsRNA called short interfering RNA or siRNA. Remarkable Properties of RNAi dsRNA (not ssRNA) is interfering agent Sequence-specific loss of mRNA and protein Effective against exons not introns Potent (few dsRNA molecules are effective) Persistent (affects next generation) Effects can cross cell barriers (feed, soak) Mechanism of RNAi Two step mechanistic model: Initiation step Generation of siRNAs Effective step Degradation of target mRNA Amplified mechanistic : 1. Primate siRNA- dsRNAs were cut by Diser 2. Sub siRNA- produced dsRNAs by RdRP were cut Diser into new siRNA Initiation Step ATP ATP ADP + ppi ADP + ppi DICER KINASE RdRP Effective Step RISC Genome-wide RNAi screens Invertebrates have proved useful in high-throughput RNAi screens. Apply global RNAi screens to ageing-related disorders are likely to be of considerable benefit. RNAi screens is high specificity,but must be confirmed using complementary techniques. Gene silencing Gene silencing is nature response to invasive virus in the plant-RNA-mediated anti-virus immunoreaction. transcriptional gene silencing,TGS post- transcriptional gene silencing, PTGS RNAi? Long dsRNA induces non-specific response of mammalian 21nt