研究生分子生物学工作基础新基因研究路线2

Loss of Function Inhibition of activityInhibition of expressionInhibition of Activityl Specific inhibitorsl Dominant negativesl Specific antibodiesHistone Deacetylase (HDAC) inhibitorsHistone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nature Reviews Cancer. 2006 Vol 6. 38-51Dominant Negative A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell. This usually occurs if the product can still interact with the same elements as the wild-type product, but block some aspect of its function. Examples: 1. A mutation in a transcription factor that removes the activation domain, but still contains the DNA binding domain. This product can then block the wild-type transcription factor from binding the DNA site leading to reduced levels of gene activation.2. A protein that is functional as a dimer. A mutation that removes the functional domain, but retains the dimerization domain would cause a dominate negative phenotype, because some fraction of protein dimers would be missing one of the functional domains.Dominant negative mutant of a transcription factorpol IIP P PInhibition of Expressionl RNA interference (RNAi)l Knockout RNA INTERFERENCE (RNAi)Andrew Z. Fire and Craig C. Mello“for their discovery of RNA interference - gene silencing by double-stranded RNA“. Nobel Prize in Physiology and Medicine 2006Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver, Craig C. Mello. Potent and specic genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391: 806 811, 1998 RNAi in Mammalian cells 30 base pair double-stranded RNAShort interfering RNAs 30 base pairsPKRinactivePKRactive2, 5-ASinactive2, 5-ASactiveDegradation of mRNA(Sequence-specific effects)(Global effects)CleavageInhibition of protein synthesis Degradation of mRNAsRNAiEndogenous SRC-1 RNAi5 C C U C A G G G C A G A G A A C C A U C U T TT T G G A G U C C C G U C U C U U G G U A G A 51 943 963 4365SRC-1 mRNASmall interfering RNA (siRNA)AIB1 GRIP1 Lamin ALamin CSRC-1 siRNALamin siRNABufferSRC-1SRC-1 siRNALamin A siRNABufferBufferb-gallacZSRC-1 siRNALamin siRNABuffer SRC-1 siRNALamin siRNABuffer SRC-1 siRNALamin siRNABuffer- + + +Shang, Y., and Brown, M. Science 295: 2465-2468, 2002Mechanisms Involved in RNAi RNase III family members are among the few nucleases that show specificity for dsRNA. Analysis of the Drosophila and C. elegans genomes reveals 3 types of RNase III enzymes. 1. the canonical RNase III, which contains a single RNase III signature motif and a dsRNA-binding domain (dsRBD; for example RNC_CAEEL ).2. Drosha, a Drosophila enzyme that contains two RNase III motifs and a dsRBD. 3. Homeless contains two RNase III signatures and an amino-terminal helicase domain (Drosophila CG4792 and CG6493; C. elegans K12H4.8)The Discovery of DicerDicer Family Drosophila: Dicer C. elegans: K12H4.8 Arabidopsis: CARPEL FACTORY, T25K16.4 and AC012328_1) Mammals: Helicase-MOI Schizosaccharomyces pombe: YC9A_SCHPO RNAi an ATP-dependentProcess Based upon the known mechanisms for the RNase III family of enzymes, Dicer is thought to work as a dimeric enzyme. Cleavage into precisely sized fragments is determined by the fact that one of the active sites in each Dicer protein is defective, shifting the periodicity of cleavage from 911 nucleotides for bacterial RNase III to 22 nucleotides for Dicer family members. Methods of RNAi knockdown in mammalian cellsSchematic of the siRNA mediated RNA interference pathway. After entry into the cytoplasm, siRNA is either loaded onto RISC directly or utilize a Dicer mediated process. After RISC loading, the passenger strand departs, thereby commencing the RNA interference process via target mRNA cleavage and degradation. siRNA loaded RISCs are also found to be associated with nucleolus region and maybe shuttled in and out of nucleus through an yet unidentified process.siRNA vs. shRNA: Similarities and differences. Advanced Drug Delivery Reviews 61 (2009) 746759Schematic of the shRNA mediated RNA interference pathway. After delivery of the shRNA expression vector into the cytoplasm, the vector needs to be transported into the nucleus for transcription. The primary transcripts (pre-shRNA) follow a similar route as discovered for the primary transcripts of microRNA. The primary transcripts are processed by the Drosha/DGCR8 complex and form pre shRNAs. Pre-shRNAs are transported to the cytoplasm via exportin 5, to be loaded onto the Dicer/TRBP/PACT complex where they are further processed to mature shRNA. Mature shRNA in the Dicer/TRBP/PACT complex are associated with Argonaute protein containing RISC and provide RNA interference function either through mRNA cleavage and degradation, or through translational suppression via p-bodies.The Specificity of RNAi if siRNAs elicit a specific response, then all of the siRNAs designed against the same target would be expected to produce similar gene expression signatures the siRNAs designed against different target genes would be expected to show different gene expression signatures Important Parameters in siRNA Transfection Experiments Health of cultured cells Transfection method Adherent mammalian cells have been traditionally pre-plated into tissue culture wells and allowed to attach, recover, and grow for 24 h prior to transfection. Reverse transfection is an alternative method of transfection where cells are transfected while still in suspension (i.e. after trypsinization and prior to plating). Reverse transfection is believed to increase cell exposure to transfection complexes often leading to greater transfection efficiency. Transfection conditions Length of cell exposure to transfection agents should be optimized to minimize cellular toxicity and maximize siRNA activity by varying the amount of transfection agent and cell exposure time to transfection complexes. Quality and quantity of siRNA/site/cn/zh/home/References/Ambion-Tech-Support/rnai-sirna/tech-notes/optimizing-sirna-transfection-for-rnai.htmlSummary of Different siRNA Delivery MethodsGene Knockout (9-12 months) Cloning and mapping of mouse genomic target DNA from a mouse cDNA sequence (3-5 weeks) Designing and creating a targeting vector (6-10 weeks) Electroporation and selection of ES cells (6-10 weeks) Identification of homologous recombinant ES clones (2 weeks) Expansion of recombinant clones (1 weeks) Blastocyst injection of recombinant ES clones (4-6 weeks) Identification of germline transmission