How To Analyze A New Gene


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How To Analyze A New Gene

  1. 1. How to analyze a new gene A novel gene co 物 o. 生 m io 做 bb 心 Gene expression Gene Functions w. -专 ww 秀 物 Linkage analysis 生 Protein Location and complex Biochemical and biological activity Domain analysis and structure determination
  2. 2. Protein Expression is regulated at multiple levels Gene copy number DNA Chromosome structure Methylation and acetylation Transcription Promoter activity co 物 Inducible transcription factor o. 生 Alternative splicing RNA m io 做 mRNA transport and stability bb 心 Ribosome binding site w. -专 Translation Codon usage Termination ww 秀 Protein folding and targeting 物 Protein Protein stability 生 Phosphorylation Post-translational Glycosylation modification Ubiquitination Sumonylation Palmolylation … Biological Cellular process Activity
  3. 3. Methods for Analysis of Gene Expression Analyzing promoter: Analyzing transcription: --- Nuclear Run-on assay --- Northern blots co 物 --- DNA Foot-printing assay --- RNase protection assay o. 生 --- EMSA --- Reverse transcription PCR m io 做 --- Reporter gene assay --- Real Time PCR --- DNA truncation and --- In situ hybridization bb 心 site-directed mutagenesis --- Primer extention assay w. -专 --- in vitro reconstitution assay --- CHIP assay ww 秀 物 生 Comparing transcriptomes: Translational analysis: --- Differential screening --- Western Blots --- Subtractive hybridization --- Immunocytochemistry --- Differential display Immunohistochemistry --- Array-based methods --- Protein modification --- Proteomics
  4. 4. Topics covered in this lecture Promoter analysis co 物 o. 生 m io 做 Western Blot bb 心 w. -专 RT-PCR ww 秀 物 生 Real Time PCR RNA interference (RNAi)
  5. 5. Promoter analysis co 物 Promoters: DNA element(s) or sequence which determines o. 生 the site of transcription initiation of RNA polymerase and m io 做 bb 心 may also affect the efficiency of initiation w. -专 Promoters are and should be defined functionally as ww 秀 sequences that direct transcription initiation in vivo or in vitro 物 using a functional assay (cause transcription of an RNA). 生
  6. 6. Components of a typical gene involved in gene regulation Matrix Attachment region Matrix Attachment region co 物 o. 生 Boundary element Boundary element m io 做 bb 心 Gene w. -专 Regulatory promoter ww 秀 Enhancer 物 生 DNA -500 -4000 -40 +50 Core promoter
  7. 7. Sequence elements in a typical core promoter co 物 o. 生 Downstream core Promoter element TATA motif Initiator m io 做 bb 心 Py Py AN T/A Py Py R G A/T C G T G TATAAA G/C G/C G/C CGCC w. -专 -38 -32 -26 +1 +30 ww 秀 TF II D 物 生 TF II B TF II A Polymerase II
  8. 8. A simplified model of enhanceosome formation co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  9. 9. Strategy for promoter analysis Isolate putative promoter from genomic clone co 物 o. 生 Sequence putative clone m io 做 Determine transcription start site bb 心 w. -专 Establish functional assays ww 秀 Identify cis-acting DNA elements and trans-acting factors 物 生 Determine relevance of these factors Establish regulatory model of the promoter in vivo and in vitro
  10. 10. Nuclear Run-on Transcription Assay ---An indirect measure of the in vivo rate of transcription initiation from a given gene ---The amount of specific radiolabeled RNA observed is approximately proportional to the number of polymerase molecules associated with the gene when the cells were lysed and chilled, which in turn is proportional to the rate of transcription initiation from the gene. co 物 o. 生 Add Hybridize specific Isolate NTPs Incubate radiolabeled RNA m io 做 Radio- and at 37 Lyse cells on ice Pellet nuclei to unlabeled cDNA labeled Buffer for several immobilized on bb 心 RNA (One minutes filter labeled) w. -专 ww 秀 Little or no transcription of gene Uninduced cells 物 生 (e.g plasmid backbone) Induced cells Negative control gene of interest Positive control Active transcription (e.g β-actin) cDNA from
  11. 11. Primer Extension assay 5‘ 3‘ (20-25 bases) Synthesize primer and label at co 物 5’ end with [γ-32p]ATP and T4 polynucleotide kinase o. 生 5‘ 3‘ m io 做 Hybridize hot primer to specific mRNA bb 心 mRNA 50-150 bases Extended primer w. -专 5‘ 3‘ 3‘ 5‘ Extend primer to 5’ end ww 秀 of mRNA using reverse transcriptase G A T C 物 mRNA 5‘ 3‘ 生 cDNA 3‘ 5‘ Analyze radiolabeled DNA on sequencing gel Free excess primer
  12. 12. RNase protection assay +1 +1 Screen genomic DNA library to +59 +59 -123 -123 find the DNA fragment spanning HindIII Sub-clone Hind III co 物 Sp6 promoter Sp6 promoter the region thought to contain the o. 生 transcription start site for the gene of interest m io 做 bb 心 t en m w. -专 g ra e tf Sp6 RNA poltmerase ob en pr 3‘ NTPs (α-32P)UTP ist d ww 秀 ste es er ge as i nd 物 G A T C mRNA RN U 生 5‘ 3‘ 5‘ 3‘ 5‘ +1 +59 +1 +59 -123 -123 +1 +59 RNase T1 Hybridize and/or RNase A
  13. 13. 5‘ RACE Procedure (Rapid Amplification of cDNA Ends) 3‘ mRNA 5‘ AAAAA co 物 3‘ mRNA Hybridize primer 100-200 bases 5‘ o. 生 AAAAA from 5’ end of mRNA 3‘ 5‘ m io 做 bb 心 3‘ mRNA 5‘ Reverse Transcription AAAAA w. -专 3‘ 5‘ ww 秀 Ligate oligo of known sequence 3‘ mRNA to 3’ end using RNA ligase or 5‘ 物 AAAAA Extend cDNA using terminal 生 5‘ 3‘ transferase(TdT) and dGTP PCR 5‘ 3‘ Insert PCR produt into vector Sequence individual clones
  14. 14. Establishing functional assays for promoter analysis co 物 o. 生 Transient transfection assay m io 做 bb 心 Stable Transfection assay w. -专 ww 秀 In vitro transcription assay 物 生 Transgenic assay Homologous recombination assay
  15. 15. Transient transfection assay Constitutive promoter (SV40, HSV-TK) Promoter of interest Reporter Reporter Reporter gene gene gene co 物 or o. 生 m io 做 Distant control bb 心 region of interest w. -专 Transfect effective and control cell (Inducible genes or cell type specific genes) ww 秀 Including normalization plasmid 物 Stimulation Grow 24-72 hours 生 Harvest cells. Prepare protein extract or mRNA Measure enzymatic activity Measure reporter of reporter gene product mRNA levels
  16. 16. Stable Transfection assay Measure enzymatic activity Promoter of reporter gene product co 物 of interest Dominant selectable o. 生 marker gene Reporter gene m io 做 Measure reporter Constitutive promoter mRNA levels bb 心 (SV40, HSV-TK) w. -专 ww 秀 Transfect cultured cells 物 生 Drug selection for 1-4 weeks Harvest cells. Expand several cell clones or Prepare protein extract or mRNA pools of drug resistant cells
  17. 17. In vitro transcription assay Promoter A 360-bp sequence devoid of G of interest residues on the noncoding strand. G-less cassette The resulting message is G-less. co 物 Transcription should in principle o. 生 terminate at the end of the G-less m io 做 cassette. bb 心 w. -专 ww 秀 Incubate plasmid in nuclear extract 物 with ATP, CTP, [32P]UTP In crude extract, despite extensive dialysis, 生 Including 3’-O-Methyl-GTP there are enough contaminating GTP to allow nonspecific RNA synthesis. Acting as chain terminator Gel electrophoresis analysis
  18. 18. Choice of Reporter genes CAT (chloramphenicol acetyltransferase) Transfers radioactive 14C acetyl groups to chloramphenicol. detection by thin layer chromatography and autoradiography co 物 GAL (β-galactosidase) o. 生 Hydrolyzes colourless galactosides to yield coloured products. Assay m io 做 change/production of colour bb 心 SEAP (secreted human placental alkaline phosphatase) w. -专 highly-sensitive bioluminescent alkaline phosphatase assay ww 秀 LUC (luciferase) 物 Oxidizes a luciferin emitting photons. Count photons by luminometer or photon- 生 counting camera. Different luciferases avaiable GFP (green fluorescent protein) Fluoresces on irradiation with UV – observed upon fluorescence microscopy or measure fluorescence. Use destabilised forms
  19. 19. Common transfection methods Calcium phosphate: DNA is mixed with calcium chloride in a phosphate buffer, resulting in the formation of a DNA-calcium phosphate precipitate, which is layered on cells and taken up by endocytosis. co 物 For both transient and stable transfection of adherent cells o. 生 DEAE-dextran: m io 做 The soluble complex between negatively charged DNA and cationic DEAE-dextran is bb 心 taken up by endocytosis into cells. For transient transfection of both adherent and non-adherent cells. w. -专 Rarely useful for stable transfection because of toxicity. ww 秀 Electroporation: 物 A high voltage electroshock induces or stabilizes pores in the plasma membrane through 生 which the DNA enters the cell. For cells resistant to transfection Lipofection: Positively charged cationic lipid compounds Exceptionally high efficiency for some cell line Polycations:
  20. 20. co 物 o. 生 m io 做 bb 心 Warming up the Basic PCR procedure w. -专 ww 秀 物 生
  21. 21. Dissection of promoter cis-elements (DNA motif mapping) Identification of cis-elements using in vivo or in vitro co 物 protein-DNA interaction methods o. 生 --- EMSA m io 做 --- DNase Footprinting bb 心 w. -专 Identification of cis-elements by database analysis ww 秀 --- 物 生 Identification of cis-elements by comprehensive mutant analysis --- Deletion analysis --- Generation of internal deletion mutants --- Generation of substitution mutants (Linker scanning) --- Site-directed mutagenesis
  22. 22. Deletion analysis Long Promoter Reporter of interest gene co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 PCR or Restriction Enzyme blunt ligation 物 Reporter gene activity 生
  23. 23. Generation of internal deletion mutants by PCR +1 5‘ 3‘ 3‘ 5‘ PCR co 物 5‘ 3‘ o. 生 3‘ 5‘ 5‘ 3‘ m io 做 3‘ 5‘ Denature and anneal bb 心 w. -专 3‘ 5‘ 3‘ 5‘ Fill in with Taq and dNTPs ww 秀 物 3‘ 5‘ 3‘ 5‘ 生 PCR +1 5‘ 3‘ 3‘ 5‘ Clone into reporter plasmids and carry out reporter assay
  24. 24. Generation of substitution mutants by PCR (Linker scanning) +1 5‘ 3‘ 6-10 base pairs 3‘ 5‘ Same length Different sequence co 物 PCR o. 生 5‘ 3‘ 3‘ m 5‘ io 做 5‘ 3‘ 3‘ 5‘ bb 心 Denature and anneal w. -专 3‘ 5‘ 3‘ 5‘ ww 秀 Fill in with Taq and dNTPs 物 3‘ 5‘ 生 3‘ 5‘ PCR +1 5‘ 3‘ 3‘ 5‘ Clone into reporter plasmids and carry out reporter assay
  25. 25. Site-directed mutagenesis Pfu turbo DNA polymerase co 物 o. 生 m io 做 bb 心 w. -专 Methylated nucleotide Digest the methylated ww 秀 parental DNA by DpnI 物 生 Transform the circular, nicked dsRNA into E.coli.. The bacteria will repair the nicks in the mutated DNA
  26. 26. Filter Blotting 1979: Towbin Dot blot or Slot blot co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 ECL detection of 生 human serum transferrin --- Providing quantitative information about protein expression levels --- Useful for on multiple samples performed in parallel --- Lacking information on protein molecular weight, its isoforms or post-translational modification
  27. 27. Western Blotting Protein mixture co 物 o. 生 m io 做 SDS-PAGE bb 心 w. -专 ww 秀 Blot 物 生 ECL detection of human serum transferrin Detection
  28. 28. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  29. 29. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  30. 30. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  31. 31. PVDF membranes offer better protein retention, physical strength and chemical compatibility co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  32. 32. Pretreatment of PVDF membrane PVDF is an inherently hydrophobic polymer and will not wet-out in co 物 aqueous solutions. In order for a PVDF membrane to be compatible o. 生 with aqueous systems, it must first be wet in a 50% (v/v) or m io 做 greater concentration of alcohol,methanol,or isopropanol. Complete bb 心 wetting is evident by a change in the membrane’s appearance from w. -专 opaque to semi-transparent. ww 秀 The alcohol is then removed from the membrane by extensive rinsing 物 in water, and the membrane is equilibrated in the appropriate 生 buffer. Once the membrane is wet, protein binding can be achieved by simply bringing the protein into contact with the membrane.
  33. 33. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  34. 34. Tank Transfer System co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 Tris/glycine transfer buffer: The methanol added to transfer buffers has two major functions: 25 mM Tris base --- Stabilizes the dimensions of the gel 192 mM glycine, --- Strips complexed SDS from the protein molecules 10% (v/v) methanol pH 8.3
  35. 35. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Semi-dry Transfer System
  36. 36. Electrotransfer Timing: the longer is not always the better co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 12 hours 1.5 hours 12 hours 1.5 hours Coomassie Blue Staining
  37. 37. co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 Long transfer times are best suited for tank systems, which normally require cooling of the unit and internal recirculation of the transfer buffer. In semi-dry transfer, however, prolonged blotting may result in buffer depletion, overheating and gel drying.
  38. 38. Protein Visualazation on blot co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  39. 39. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Pre-stained Ladder
  40. 40. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Protein Identification
  41. 41. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  42. 42. Chromogenic Detection: Coupled enzyme (Alkaline phosphatase, AP) catalyzes a reaction resulting in the deposit of an insoluble colored precipitate. co 物 (BCIP: 5-bromo-4-chloro-3-indolylphosphate o. 生 NBT: nitroblue tetrazolium salt) m io 做 bb 心 w. -专 ww 秀 物 生
  43. 43. Chemiluminescent Detection: Coupled enzyme(horseradish peroxidase,HRP) catalyzes a reaction that results in the production of visible light. co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 Fluorescent Detection:
  44. 44. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  45. 45. 生 50OC/30 min 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  46. 46. Reverse Transcription-PCR Flow Chart (RT-PCR) co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  47. 47. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Isolating RNA for RT-PCR
  48. 48. Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-chloroformExtraction cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysate by extraction with phenol:chloroform at reduced pH. Many samples can be processed co 物 simultaneously and speedily. The yield of total RNA depends on the tissue or cell resource and is generally in the range of 4-7 µg/ml starting tissue or 5-10 µg/106 cells. o. 生 m Prepare all reagents with DEPC-treated H2O. io 做 bb 心 Guanidnium thiocyanate is a strong denaturing agent, preventing RNA degradation w. -专 Proteinase K degrades protein contaimination RNAase-free DNAase degrades DNA. ww 秀 物 Acidic pH Neutral pH 生 Aqueous layer Aqueous layer ( RNA) (DNA and RNA) Protein precipitate Protein precipitate Phenol layer Phenol layer (DNA)
  49. 49. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Getting Rid of DNA
  50. 50. Column-base RNA purification co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  51. 51. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  52. 52. Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography co 物 o. 生 m io 做 Chromatography on oligo(dT) columns is the preferred method for large- bb 心 scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian w. -专 cells. A combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. Typically, between 1% and 10% of ww 秀 the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA. 物 生
  53. 53. Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  54. 54. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  55. 55. 生 物 ww 秀 RT-PCR w. -专 bb 心 io 做 o. 生 co 物 m RT
  56. 56. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m Relative RT-PCR
  57. 57. Determining Exponential Phase co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  58. 58. The Evolution of PCR to Real-Time PCR co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 PCR has completely revolutionized the detection of RNA and DNA. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring.
  59. 59. Limitations of Traditional PCR co 物 Poor Precision o. 生 Low sensitivity m io 做 bb 心 Short dynamic range < 2 logs w. -专 Low resolution ww 秀 Non - Automated 物 Size-based discrimination only 生 Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing
  60. 60. What is Real-Time PCR co 物 A PCR system in which we can monitor the amplification reaction o. 生 as it is occuring. It incorporates the ability to directly measure and m io 做 quantify the reaction while amplification is taking place. bb 心 w. -专 ww 秀 物 生
  61. 61. co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 A basic PCR run can be broken up into three phases: 生 Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Linear (High Variability): The reaction components arebeing consumed, the reaction is slowing, and products are starting to degrade. Plateau : The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
  62. 62. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  63. 63. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  64. 64. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  65. 65. The Threshold line is the level of threshold detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of co 物 the amplification for the most accurate o. 生 reading. The cycle at which the m sample reaches this level is called the io 做 Cycle Threshold, Ct. bb 心 w. -专 ww 秀 物 生
  66. 66. Common Standards for Real Time PCR co 物 o. 生 --- Glyceraldehyde-3-phosphate dehydrogenase mRNA m io 做 bb 心 --- Beta-actin mRNA w. -专 --- MHC I (major histocompatability complex I) mRNA ww 秀 --- Cyclophilin mRNA 物 --- mRNAs for certain ribosomal proteins RPLP0 生 28S or 18S rRNA
  67. 67. REAL TIME PCR co 物 • kinetic approach o. 生 • early stages m io 做 bb 心 • while still linear w. -专 ww 秀 物 生
  68. 68. co 物 3. 5. ccd o. 生 intensifier detecto m io 做 1. halogen r tungsten lamp bb 心 2b. emission 350,00 filters 0 pixels w. -专 ww 秀 物 2a. excitation 生 filters 4. sample plate
  69. 69. The 5’ exo-nuclease activity of AmpliTaq® Polymerase and FRET (Fluorescent Resonant Energy Transfer) makes it possible to detect PCR amplification in Real-Time. co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  70. 70. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  71. 71. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  72. 72. co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 The Scorpions uni-probe consists of a single-stranded bi-labeled fluorescent probe sequence held in a hairpin-loop conformation (approx. 20 to 25 nt) by complementary stem sequences (approx. 4 to 6 nt) 物 on both ends of the probe. The probe contains a 5’end reporter dye and an internal quencher dye 生 directly linked to the 5’end of a PCR primer via a blocker. The blocker prevents Taq DNA polymerase from extending the PCR primer. The close proximity of the reporter dye to the quencher dye causes the quenching of the reporter's natural fluorescence. At the beginning of the real-time quantitative PCR reaction, Taq DNA polymerase extends the PCR primer and synthesizes the complementary strand of the specific target sequence. During the next cycle, the hairpin-loop unfolds and the loop- region of the probe hybridizes intramolecularly to the newly-synthesized target sequence. The reporter is excited by light from the real-time quantitative PCR instrument (hg ). Now that the reporter dye is no longer in close proximity to the 1 quencher dye, fluorescence emission may take place (hg ).
  73. 73. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  74. 74. SYBR Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR Green is a dye that binds the Minor Groove of double stranded DNA. When SYBR Green dye co 物 binds to double stranded DNA, the o. 生 intensity of the fluorescent emissions m io 做 increases. As moredouble stranded amplicons are produced, SYBR Green bb 心 dye signal will increase. w. -专 ww 秀 物 生
  75. 75. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  76. 76. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  77. 77. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  78. 78. RNA interference (RNAi) post-transcriptional gene silencing (PTGS) Antisense-mediated silencing: large amounts of a nucleic acid whose sequence is complementary to the target messenger RNA are delivered into the cytoplasm of a cell. Base pairing between the 'sense' mRNA sequence and the complementary 'antisense' interfering nucleic acid is thought to passively block the processing or co 物 translation of mRNA, or result in the recruitment of nucleases that promote mRNA o. 生 destruction. m io 做 Unexpected results: bb 心 ---Both the antisense and the control sense RNA preparations induced silencing of C. w. -专 elegans par-1 mRNA to block par-1 expression. ---Silencing effect could be transmitted in the germ line. ww 秀 ---The silencing effect could also spread from tissue to tissue within the injected 物 animal. 生 Hypothesis: ---The properties of RNAi demands the existence of cellular mechanisms that initiate and amplify the silencing signal, and suggest that the RNAi mechanism represents an active organismal response to foreign RNA. ---dsRNA, often encountered by cells during viral infection, might itself be the initial trigger. dsRNA proved to be an extremely potent activator of RNAi — at least 10- fold and perhaps 100-fold more effective than purified preparations of single-stranded RNA.
  79. 79. Natural mechanism of RNA interference co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生 Dicer: RNase III enzyme RISC: RNA-induced silencing complex
  80. 80. Advantages and Disadvantages of Different Gene Suppression Strategies co 物 Advantage Disadvantage o. 生 m io 做 Transfection-dependent RNAi Potent, specific “Knockdown” not bb 心 “knockout” w. -专 Antisense DNA Variable efficacy and specificity Simple, inexpensive oligonucleotides Transfection-dependent ww 秀Ability to determine functions of dominant negative Transfection-dependent 物 discrete regions of a protein Unanticipated side effects mutant gene 生 Stable suppression possible Small molecule Often nonspecific Ease of administration May not be available inhibitors Laborious to produce Expensive “Knockout mouse” 100% gene silencing Lethal mutants may prevent embryonic development
  81. 81. siRNA design There are expanding libraries of validated, commercially available siRNAs directed toward some commonly co 物 targeted genes. These may be of use, if available. However, o. 生 m io 做 if the gene of interest has not been targeted using siRNA bb 心 before, a novel siRNA must be developed. w. -专 ww 秀 物 Selection of the targeted region is currently a trial-and- 生 error process, but with the likelihood of 80–90% success , given a large enough random selection of target genes
  82. 82. Methods to generate short RNAs that silence gene expression Silencing by RNAs that are generated in vitro co 物 o. 生 m io 做 bb 心 w. -专 Silencing by RNAs that ww 秀 are generated in vivo 物 (H1 and U6) 生
  83. 83. Advantages and Disadvantages of Different siRNA Delivery Strategies Advantage Disadvantage co 物 o. 生 Rapid synthesis Transient RNAi Chemical and in vitro m io 做 High purity using chemical enzymatic synthesis expensive for multiple siRNA bb 心 synthesis w. -专 More economical for More labor intensive to multiple sequences DNA plasmid vector or ww 秀 generate cassette Stable RNAi achievable using 物 Transfection-dependent selection marker 生 May be effective in cells resistant to transfection with More labor intensive to dsRNA and plasmids generate Virus-mediated Integration produces stable Potential biohazard RNAi even in the absence of a selection pressure
  84. 84. Selection of siRNA duplexes co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 [1]Select the target region from the open reading frame of a given cDNA sequence preferably 生 50 to 100 nt downstream of the start codon. Avoid 5′ or 3′ untranslated regions (UTRs) or regions close to the start codon as these may be richer in regulatory protein binding sites. [2]Search for sequences 5′-AA(N19)UU . Choose those with approximately 50% G/C content ( from 32 to 79% G/C content ). If not, follow (B). [3] Blast-search the selected siRNA sequences against EST libraries or mRNA sequences of the respective organism to ensure that only a single gene is targeted. [4] It may be advisable to synthesize several siRNA duplexes to check the silencing effectivity. A nonspecific siRNA duplex is needed as control.
  85. 85. Preparation of siRNA duplexes by Chemical Synthesis co 物 Proligo (Hamburg, Germany, o. 生 Dharmacon Research (Lafayette, CO, m Pierce Chemical ( io 做 Glen Research (Sterling, VA, bb 心 ChemGenes (Ashland, MA, w. -专 Cruachem (Glasgow, UK, ww 秀 物 生 Analysis of siRNA duplex formation
  86. 86. Schematic for a typical RNAi experiment using chemically synthesized siRNA co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生
  87. 87. Silencing of a green fluorescent protein (GFP) reporter in C. elegans co 物 o. 生 RNAi Control m io 做 bb 心 w. -专 ww 秀 物 生
  88. 88. 生 物 ww 秀 19 bases w. -专 bb 心 io 做 o. 生 co 物 m Vector Based RNAi
  89. 89. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  90. 90. 生 物 ww 秀 w. -专 bb 心 io 做 o. 生 co 物 m
  91. 91. Silencing of the cytoskeletal intermediate filament protein vimentin co 物 o. 生 m io 做 bb 心 w. -专 A B ww 秀 Longer 物 exposure 生
  92. 92. Silencing of SV40 large T antigen in a stably transformed rat fibroblast cell line co 物 o. 生 m io 做 bb 心 w. -专 ww 秀 物 生