Graduate Course For Year October_13_ 2005

基因表达调控的研究策略和常用方法

王

琛

分子细胞重点实验室 中科院上海生物化学与细胞生物学研究所

SIBCB_Chen Wang

How to analyze a new gene A novel gene

Gene expression

Gene Functions

Linkage analysis Protein Location and complex Biochemical and biological activity Domain analysis and structure determination

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Combinations of a few gene regulatory proteins can generate many different cell types during development

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Protein Expression is regulated at multiple levels DNA Transcription

Promoter activity Inducible transcription factor

RNA

Alternative splicing mRNA transport and stability Ribosome binding site Codon usage Termination

Translation

Protein

Protein folding and targeting Protein stability Phosphorylation Glycosylation Ubiquitination Sumonylation Palmolylation …

Post-translational modification

Biological Activity SIBCB_Chen Wang

Gene copy number Chromosome structure Methylation and acetylation

Cellular process

Methods for Analyzing Gene Expression Analyzing transcription:

Analyzing promoter:

--- Northern blots --- RNase protection assay --- Reverse transcription PCR --- Real Time PCR --- In situ hybridization --- Primer extention assay

--- Nuclear Run-on assay --- DNA Foot-printing assay --- EMSA --- Reporter gene assay --- DNA truncation and site-directed mutagenesis --- in vitro reconstitution assay --- CHIP assay

Comparing transcriptomes:

Translational analysis:

--- Differential screening --- Subtractive hybridization --- Differential display --- Array-based methods

--- Western Blots --- Immunocytochemistry Immunohistochemistry --- Protein modification --- Proteomics

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Topics covered in this lecture Promoter and Enhancer analysis Western Blot RT-PCR Real Time PCR RNA interference (RNAi) SIBCB_Chen Wang

Promoter analysis Promoters: DNA element(s) or sequence which determines the site of transcription initiation of RNA polymerase and may also affect the efficiency of initiation Enhancer: control element that elevates the levels of transcription from a promoter, independent of orientation or distance Promoters and Enhancers are and should be defined functionally as sequences that direct or promote transcription initiation in vivo or in vitro using a functional assay (cause transcription of an RNA). SIBCB_Chen Wang

Enhancer

Regulatory promoter

DNA -4000

-500

-40 +50

Core promoter

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Matrix Attachment region

Gene

Boundary element

Boundary element

Matrix Attachment region

Components of a typical gene involved in gene regulation

Sequence elements in a typical core promoter

TATA motif G/C G/C G/C CGCC

-38 TF II D TF II B TF II A Polymerase II

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-32

Initiator Py Py AN T/A Py Py

TATAAA

-26

+1

Downstream core Promoter element R G A/T C G T G

+30

A simplified model of enhanceosome formation

CBP c-Jun

TAFs

ATF-2 Pol II IRF3

TAFs

p300 IRF7

II B

IRF3

II F

NF-κB II A

TBP TATA

Enhancer

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II E II H

Core promoter

Strategy for promoter analysis Isolate putative promoter from genomic clone Sequence putative clone Determine transcription start site Establish functional assays Identify cis-acting DNA elements and trans-acting factors Determine relevance of these factors Establish regulatory model of the promoter in vivo and in vitro

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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. Hybridize specific radiolabeled RNA to unlabeled cDNA immobilized on filter

Active transcription

Negative control (e.g plasmid backbone)

Little or no transcription of gene

Induced cells

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Isolate Radiolabeled RNA

Positive control (e.g β-actin)

Uninduced cells

Pellet nuclei

Incubate at 37 for several minutes

cDNA from gene of interest

Lyse cells on ice

Add NTPs and Buffer (One labeled)

DNA Sequencing

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Primer Extension assay 5‘

3‘ (20-25 bases)

Synthesize primer and label at 5’ end with [γ-32p]ATP and T4 polynucleotide kinase 5‘

3‘

5‘

3‘

Extend primer to 5’ end of mRNA using reverse transcriptase

mRNA

50-150 bases

3‘

5‘

G

mRNA

5‘ 3‘

5‘

A

T

C

Extended primer

Hybridize hot primer to specific mRNA

3‘

cDNA

Analyze radiolabeled DNA on sequencing gel Free excess primer SIBCB_Chen Wang

RNase protection assay +1 +59

-123 Sub-clone Hind III

+59

-123

HindIII

Sp6 promoter

Sp6 promoter

G

A

T

C

U nd ig es te d pr RN ob as e er es ist en tf ra gm

en t

Screen genomic DNA library to find the DNA fragment spanning the region thought to contain the transcription start site for the gene of interest

+1

3‘

Sp6 RNA poltmerase NTPs (α-32P)UTP

mRNA 5‘ 3‘ 3‘

+1

5‘ -123

+1

+59

-123

+59 RNase T1 and/or RNase A

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5‘

Hybridize

+1

+59

5‘ RACE Procedure (Rapid Amplification of cDNA Ends) 5‘

mRNA

3‘ AAAAA

5‘

mRNA

3‘ AAAAA

3‘

5‘ 3‘ AAAAA

mRNA

5‘ 3‘

Hybridize primer 100-200 bases from 5’ end of mRNA

Reverse Transcription

5‘ 3‘ AAAAA

mRNA

5‘

5‘

3‘

3‘

5‘

Ligate oligo of known sequence to 3’ end using RNA ligase or Extend cDNA using terminal transferase(TdT) and dGTP PCR

Insert PCR produt into vector Sequence individual clones SIBCB_Chen Wang

Establishing functional assays for promoter and enhancer analysis Transient transfection assay Stable Transfection assay In vitro transcription assay Transgenic assay Homologous recombination assay

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Transient transfection assay Constitutive promoter (SV40, HSV-TK)

Promoter of interest Reporter gene

Reporter gene

Reporter gene

or Distant control region of interest

Transfect effective and control cell (Inducible genes or cell type specific genes) Including normalization plasmid Stimulation

Grow 24-72 hours

Harvest cells. Prepare protein extract or mRNA Measure enzymatic activity of reporter gene product SIBCB_Chen Wang

Measure reporter mRNA levels

Dominant selectable marker gene

Reporter gene

Constitutive promoter (SV40, HSV-TK)

Transfect cultured cells

Measure reporter mRNA levels

Promoter of interest

Measure enzymatic activity of reporter gene product

Stable Transfection assay

Drug selection for 1-4 weeks Expand several cell clones or pools of drug resistant cells SIBCB_Chen Wang

Harvest cells. Prepare protein extract or mRNA

In vitro transcription assay Promoter of interest

G-less cassette

Incubate plasmid in nuclear extract with ATP, CTP, [32P]UTP Including 3’-O-Methyl-GTP

Gel electrophoresis analysis

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A 360-bp sequence devoid of G residues on the noncoding strand. The resulting message is G-less. Transcription should in principle terminate at the end of the G-less cassette.

In crude extract, despite extensive dialysis, there are enough contaminating GTP to allow nonspecific RNA synthesis. Acting as chain terminator

Transgenic or Homologous Recombination

Reporter gene

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Choice of Reporter genes CAT (chloramphenicol acetyltransferase) Transfers radioactive 14C acetyl groups to chloramphenicol. detection by thin layer chromatography and autoradiography

GAL (β-galactosidase) Hydrolyzes colourless galactosides to yield coloured products. Assay change/production of colour

SEAP (secreted human placental alkaline phosphatase) highly-sensitive bioluminescent alkaline phosphatase assay

LUC (luciferase) Oxidizes a luciferin emitting photons. Count photons by luminometer or photoncounting camera. Different luciferases avaiable

GFP (green fluorescent protein) Fluoresces on irradiation with UV – observed upon fluorescence microscopy or measure fluorescence. Use destabilised forms

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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. For both transient and stable transfection of adherent cells

DEAE-dextran: The soluble complex between negatively charged DNA and cationic DEAE-dextran is taken up by endocytosis into cells. For transient transfection of both adherent and non-adherent cells. Rarely useful for stable transfection because of toxicity.

Electroporation: A high voltage electroshock induces or stabilizes pores in the plasma membrane through which the DNA enters the cell. For cells resistant to transfection

Lipofection: Positively charged cationic lipid compounds Exceptionally high efficiency for some cell line

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PCR: DNA Copy Machine

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Dissection of promoter and enhancer (cis-elements mapping) Identification of cis-elements using in vivo or in vitro protein-DNA interaction methods --- EMSA --- DNase Footprinting

Identification of cis-elements by database analysis --- http://transfac.gbf.de/index.html

Identification of cis-elements by comprehensive mutant analysis --- Deletion analysis --- Generation of internal deletion mutants --- Generation of substitution mutants (Linker scanning) --- Site-directed mutagenesis SIBCB_Chen Wang

Deletion analysis Long Promoter of interest

PCR or

Reporter gene

Restriction Enzyme blunt ligation Reporter gene activity

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Generation of internal deletion mutants by PCR +1 5‘ 3‘

3‘ 5‘

PCR 5‘ 3‘

3‘ 5‘ 5‘ 3‘

3‘ 5‘

Denature and anneal 3‘

5‘

5‘

3‘

Fill in with Taq and dNTPs 3‘

5‘ 3‘

5‘

PCR 5‘ 3‘

+1 3‘ 5‘

Clone into reporter plasmids and carry out reporter assay SIBCB_Chen Wang

Generation of substitution mutants by PCR (Linker scanning) +1 6-10 base pairs 5‘ 3‘ Same length Different sequence

3‘ 5‘

PCR 5‘ 3‘ 5‘ 3‘

3‘ 5‘

3‘ 5‘

Denature and anneal 3‘

5‘ 3‘

5‘

Fill in with Taq and dNTPs 3‘

5‘ 3‘

5‘

PCR 5‘ 3‘

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+1 3‘ 5‘

Site-directed mutagenesis Pfu turbo DNA polymerase

Methylated nucleotide Digest the methylated parental DNA by DpnI

Transform the circular, nicked dsRNA into E.coli.. The bacteria will repair the nicks in the mutated DNA

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GENOMIC STRATEGIES TO IDENTIFY MAMMALIAN REGULATORY SEQUENCES multi-species comparative genomic sequence analysis

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Filter Blotting Blotting is a technique for visualizing a particular subset of macromolecules ( proteins, or fragments of DNA or RNA ) initially present in a complex mixture. 1979: Towbin et.al Dot blot or Slot blot

ECL detection of 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 SIBCB_Chen Wang

Western Blotting Protein mixture

SDS-PAGE

Blot ECL detection of human serum transferrin

Detection

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PVDF membranes offer better protein retention, physical strength and chemical compatibility

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Pretreatment of PVDF membrane PVDF is an inherently hydrophobic polymer and will not wet-out in aqueous solutions. In order for a PVDF membrane to be compatible with aqueous systems, it must first be wet in a 50% (v/v) or greater concentration of alcohol,methanol,or isopropanol. Complete wetting is evident by a change in the membrane’s appearance from opaque to semi-transparent. The alcohol is then removed from the membrane by extensive rinsing in water, and the membrane is equilibrated in the appropriate buffer. Once the membrane is wet, protein binding can be achieved by simply bringing the protein into contact with the membrane.

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Tank Transfer System

Tris/glycine transfer buffer: 25 mM Tris base 192 mM glycine, 10% (v/v) methanol pH 8.3 SIBCB_Chen Wang

The methanol added to transfer buffers has two major functions: --- Stabilizes the dimensions of the gel --- Strips complexed SDS from the protein molecules

Semi-dry Transfer System

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Electrotransfer Timing: the longer is not always the better

12 hours

1.5 hours

12 hours

1.5 hours

Coomassie Blue Staining

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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.

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Protein Visualization on blot

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Pre-stained Ladder

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Protein Identification

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Chromogenic Detection: Coupled enzyme (Alkaline phosphatase, AP) catalyzes a reaction resulting in the deposit of an insoluble colored precipitate. (BCIP: 5-bromo-4-chloro-3-indolylphosphate NBT: nitroblue tetrazolium salt)

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Chemiluminescent Detection: Coupled enzyme(horseradish peroxidase,HRP) catalyzes a reaction that results in the production of visible light.

Fluorescent Detection:

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50OC/30 min

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Reverse Transcription-PCR Flow Chart (RT-PCR)

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Isolating RNA for RT-PCR

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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 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. Prepare all reagents with DEPC-treated H2O. Guanidnium thiocyanate is a strong denaturing agent, preventing RNA degradation Proteinase K degrades protein contaimination RNAase-free DNAase degrades DNA. Acidic pH

Neutral pH

Aqueous layer ( RNA)

Aqueous layer (DNA and RNA) Protein precipitate Phenol layer

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Protein precipitate Phenol layer (DNA)

Getting Rid of DNA

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Column-base RNA purification

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Check the integrity of RNA extraction

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Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian cells. A combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA.

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Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase

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The necessity of RT-PCR RT-PCR

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RT

Relative RT-PCR

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Determining Exponential Phase

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The Evolution of PCR to Real-Time PCR

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.

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Limitations of Traditional PCR Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non - Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing

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What is Real-Time PCR A PCR system in which the amplification reaction was monitored as it is occuring. It incorporates the ability to directly measure and quantify the reaction while amplification is taking place.

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A basic PCR run can be broken up into three phases: 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 are being 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. SIBCB_Chen Wang

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threshold

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The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, Ct.

Common Standards for Real Time PCR --- Glyceraldehyde-3-phosphate dehydrogenase mRNA --- Beta-actin mRNA --- MHC I (major histocompatability complex I) mRNA --- Cyclophilin mRNA --- mRNAs for certain ribosomal proteins RPLP0 28S or 18S rRNA

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REAL TIME PCR • kinetic approach • early stages • while still linear

www.biorad.com SIBCB_Chen Wang

3. intensifier 1. halogen tungsten lamp

2b. emission filters

2a. excitation filters

5. ccd detector 350,000 pixels

4. sample plate

www.biorad.com SIBCB_Chen Wang

The 5’ exo-nuclease activity of AmpliTaq® Polymerase and FRET (Fluorescent Resonant Energy Transfer) makes it possible to detect PCR amplification in Real-Time.

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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) on both ends of the probe. The probe contains a 5’end reporter dye and an internal quencher dye 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 ). SIBCB_Chen Wang

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 binds to double stranded DNA, the intensity of the fluorescent emissions increases. As moredouble stranded amplicons are produced, SYBR Green dye signal will increase.

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Reverse Genetics A genetic analysis that proceeds from genotype to phenotype by gene-manipulation techniques. Advantage

Disadvantage

RNAi

Potent, specific

Transfection-dependent “Knockdown” not “knockout”

Antisense DNA oligonucleotides

Simple, inexpensive

Variable efficacy and specificity Transfection-dependent

dominant negative mutant gene

Ability to determine functions of discrete regions of a protein Stable suppression possible

Transfection-dependent Unanticipated side effects

Small molecule inhibitors

Ease of administration

Often nonspecific May not be available

100% gene silencing

Laborious to produce Expensive Lethal mutants may prevent embryonic development

“Knockout mouse” SIBCB_Chen Wang

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 translation of mRNA, or result in the recruitment of nucleases that promote mRNA destruction. Unexpected results: --- PTGS was discovered while trying to make petunias a deeper shade of purple ---Both the antisense and the control sense RNA preparations induced silencing of C. elegans par-1 mRNA to block par-1 expression. ---Silencing effect could be transmitted in the germ line. ---The silencing effect could also spread from tissue to tissue within the injected animal. SIBCB_Chen Wang

Hypothesis of RNA interference (RNAi) dsRNA, often encountered by cells during viral infection, induce robust immunological response in Mammalian organisms. 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. RNA interference has been used as a tool for reverse genetics in many different organisms including: zebrafish, planaria, hydra, fungi, Drosophila, and mammalian mouse embryo systems SIBCB_Chen Wang

Natural mechanism of RNA interference

Dicer: RNase III enzyme RISC: RNA-induced silencing complex SIBCB_Chen Wang

siRNA design There are expanding libraries of validated, commercially available siRNAs directed toward some commonly targeted genes. These may be of use, if available. However, if the gene of interest has not been targeted using siRNA before, a novel siRNA must be developed.

Selection of the targeted region is currently a trial-anderror process, but with the likelihood of 80–90% success , given a large enough random selection of target genes

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The Essence of siRNA

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Methods to Produce siRNAs Silencing by RNAs that are generated in vitro

• Chemical Synthesis • In vitro transcription • Digestion of long dsRNA by an Rnase III family enzyme (e.g. Dicer, Rnase III) •Expression in cells from an siRNA expression plasmid or viral vector • Expression in cells from a PCR-derived siRNA expression cassette

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Silencing by RNAs that are generated in vivo (H1 and U6)

Methods of siRNA delivery • Introduction of siRNA is dependant upon how it is produced and the target cell type or organism •C.elegans -- injection, soaking, or feeding •Drosophila cells -- exposure through culture medium • Mammalian cells -- transfection or electroporation

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Advantages and Disadvantages of Different siRNA Delivery Strategies Advantage

Disadvantage

Chemical and in vitro enzymatic synthesis

Rapid synthesis High purity using chemical synthesis

Transient RNAi expensive for multiple siRNA

DNA plasmid vector or cassette

More economical for multiple sequences Stable RNAi achievable using selection marker

More labor intensive to generate Transfection-dependent

Virus-mediated

May be effective in cells resistant to transfection with dsRNA and plasmids Integration produces stable RNAi even in the absence of a selection pressure

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More labor intensive to generate Potential biohazard

Selection of siRNA duplexes

[1]Select the target region from the open reading frame of a given cDNA sequence preferably 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 ). [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. SIBCB_Chen Wang

Preparation of siRNA duplexes by Chemical Synthesis Proligo (Hamburg, Germany, www.proligo.com) Dharmacon Research (Lafayette, CO, www.dharmacon.com) Pierce Chemical (www.perbio.com) Glen Research (Sterling, VA, www.glenres.com) ChemGenes (Ashland, MA, www.chemgenes.com) Cruachem (Glasgow, UK, www.cruachem.com).

Analysis of siRNA duplex formation

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Schematic for a typical RNAi experiment using chemically synthesized siRNA

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Silencing of a green fluorescent protein (GFP) reporter in C. elegans RNAi

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Control

Vector Based RNAi

19 bases

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Suppression of p53 expression Western Blot Northern Blot

Endogenous

Induced

Silencing of the cytoskeletal intermediate filament protein vimentin

A

Longer exposure

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B

Silencing of SV40 large T antigen in a stably transformed rat fibroblast cell line

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