rnai technical knock down of gene expression chul geun kim ([email protected]) laboratory of...
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RNAi RNAi
Technical Knock Down of Gene ExpressioTechnical Knock Down of Gene Expressionn
Chul Geun Kim ([email protected])
Laboratory of Molecular Genetics
Hanyang University
RNA Silencing – RNA Interference
• a new method for silencing specific genes• a potent method requiring only a few molecules per cell to be effective• a systemic method spreading out through the whole organism• to some extent hereditary and can be transmitted through the germline for several generations• epigenetic (transmission of phenotypes by mechanisms other than DNA sequence changes)
Dogmatic View of Gene Expression
DNA ProteinRNA
Post-transcriptional Control: Knock down
Quantitative Control: Levels of mRNA not proportional to levels of mRNA synthesized or protein produced
Qualitative Control: More than one protein from a single gene (e.g. Differential RNA Processing or RNA editing)
An “RNA-Centric” View of Gene Expression
RNADNA Protein
RNA: A Diverse Class of Molecules
RNADNA
Non Coding RNAs: ‘Ribo Regulators’ (~97% of RNAs Present in Human Cells are Non-Coding)
rRNAtRNA
snRNAssnoRNAsGuide RNA
Catalytic:RibozymesTelomerase
Introns5’ UTR3’ UTR
VaultY RNAs7SK
Xist, H19MicroRNAs
Viral RNAsRetrotransposons
Non Coding RNAs:
SnoRNAs • Large Family• Intron-encoded• Guide RNA Modification
Telomerase RNA • Component of telomerase• Provides template for telomere synthesis• Role in Cancer and Aging
Kill the messenger!
cap AAAA...mRNA:
in vitro transcribe an antisense RNA:
cap AAAA...
mRNA can no longer be translated into a protein
• Antisense technology was used in worms...
• Difficult to explain: sense and antisense RNA preparations are each sufficient to cause interference.
• Perhaps, the interfering RNA populations include some molecules with double-stranded character.
Synthetic Antisense Oligos
Procedure of Morphoino SynthesisProcedure of Morphoino Synthesis
Antisense Strategy
Transcription
Direct injection or ingestion by cell
Transfection of expression vector
Translation Translation BlockBlock
dsRNA-specificdsRNA-specificRNaseRNase
RNA RNA interferenceinterference
1998
1999
2000
2001
2002
1990 cosuppression of purple color in plants
dsRNA injection in worms
short RNAs identified in plants
RNAi shown in vitro
RISC activity partially purified
siRNAs identified Dicer identified
RNAi used against HIVgenome-wide RNAi screens begin
Historically Important Discoveries
RNA interference – The Beginning
- Fire et al.: "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans " Nature 391: 806-11 (1998)- Introduction of RNA into cells to interfere with function of an endogeneous gene- Investigation of the requirements for structure and delivery of interference RNA mex-3 RNA
control: not stained wt
wt + antisense RNA wt + ds RNA
• ds mixture causes potent and specific interference• ds RNA substancially more effective than antisence • effect were evident in both the injected animals and their progeny
Double-stranded RNA
inject
C. elegans
sense
antisense
RNAi in Mammalian Cells
• Long dsRNA triggers global (non-specific) gene-silencing (i.e. interferon response)• Breakthrough: Short dsRNA (~22 nt) induces RNAi
Nature 2001 411: 494-498Silencing of lamin proteins in human cells by dsRNA transfection
Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs
dsRNA
~22nt siRNAs
targetmRNA
secondary siRNAs
amplification
processing
degradation
recognition
copying+
processing
spreading
RNA Interference (RNAi)
• Double stranded RNA is responsible for post-transcriptional gene silencing of the gene from which it was derived. SPECIFIC
• NATURAL BIOLOGICAL MECHANISM IN PLANTS, INSECTS AND MAMMALS
- RNA interference (RNAi) represents an evolutionary conserved cellular defense mechanism for controlling the expression of alien genes in filamentous fungi, plants, and animals.
- dsRNA is often a byproduct of viral replication or is formed by aberrant transcription from genetic elements after random integration in the host genome.
• RNAi FUNCTIONS– regulates expression of protein coding genes (e.g. microRNA)– mediates resistance to both exogenous parasitic and exogenous pathogenic nucleic acid– response to aberrant RNAs– used experimentally to block gene expression
RNA interference – Mechanism
DICER - RNAse III, ds-specific endonuclease - Dimer, 2 catalytic domains, helicase and PAZ motif - produce 2-3 nt 3´ overhangs - ATP-dependent ribonucleaseRISC - RNA-induced silencing complex - RISC contains siRNA - precurser activated by ATP - find and destroy mRNA of complementary sequence- contains endo- and exonuclease, cleaves the hybrid in the middle followed by degradation- ARO: PAZ domain (assembly)
RNA-Mediated Gene Silencing
- RNA Interference- ‘Cosuppression’ by transgenes in plants- ‘Quelling’ in Fungi- Transcriptional Gene Silencing (TGS)
Common Trigger:
RNA-Mediated Gene Silencing
Science 2002 296:1263-1265
PTGS - Post-Transcriptional Gene Silencing
MicroRNAs (also known as Small-temporal RNAs) - let7 and lin4 (from worm) were first examples; expanding family of ‘RiboRegulators’ - negative regulator of genes - 70 nt precursor, processed by DICER, results not in dsRNA - bind target and prevent ribosomal elongation
RNAi by siRNAs Developmental regulationby stRNAs (µ RNAs)
processing~22nt
siRNAs ~22ntlin-4
processing
target recognition
target recognition
mRNA
lin-14mRNA
lin-41mRNA
3’UTR
3’UTR
degradation
~22ntlet-7
Translational repression
TGS - Transcriptional Gene Silencing
plant: - methylation in promotor regions leads to gene silencing - MET as a part of RISC
C.elegance: - polycomb-dependent mechanism - polycomb proteins ass. with RISC - chromatin remodeling: open – close transition
siRNA and Silent Chromatin - Model
- RNA homologous to centromeric repeats are processed: siRNAs
- siRNAs may recruit Clr4 histone H3 methylase
- result in methylation of H3 Lys9
- Swi6 binds chromatin
- Gene silencing
How does the RNAi machinery aid in the formation of silent chromatin?
• Possibility that siRNAs bring methyltransferases to the target loci, where they are important in histone tail modification
– i.e. Drosophila targets acteyltransferase with RNA binding chromodomain to histone H4
Related Gene Silencing Mechanisms May Function in Mammals
• X chromosome inactivation in mammals– Xist RNA coating of inactive X chromosome, but no data yet suggests that Xis
t is processed by RNAi machinery – Mouse – X inactivation and Igf2r imprinting are mediated by noncoding antisen
se RNA• Possibly in organisms with DNA methylation; Histone protein modification similar to
S. pombe would in turn cause DNA methylation and subsequent gene silencing regulation
• Future work using RNAi introduced in experiments should include study of chromatin structure or modifications at the locus of the affected gene
Science 297:1833-1837
Science 297:2215-2218
RNA interference - Mechanism
RNAi ApplicationsGENETIC TOOL
GENE THERAPY
Probing Gene Function
Combat Viral InfectionTreat Genetic Diseases (New expression strategies)
RNAi for analysis of gene function and as therapeutic - duplexes of 21-nt small interfering RNAs (siRNAs) - guide sequence-specific degradation of the homologous mRNA - degradation of targeted mRNAs, "knock-down" - targeting of essential genes causes growth arrest or triggers apoptosis
RNAi – Advantages - dsRNA is the interfering agent (stability) - it is highly specific - it is remarkably potent (only a few dsRNA molecules per cell are required for effective interference) - the interfering activity can cause interference in cells and tissues far removed from the site of introduction
siRNAs can be produced by: • Chemical synthesis • Enzymatic synthesis • RNase III/Dicer cleavage of long dsRNA • Plasmid based in vivo expression • siRNA Expression Cassettes (SECs)
siRNA Design
siRNA has UU 3′ termini; target must start with AA: 1. Scan mRNA for AA dinucleotide sequences. 2. Record the occurrence of each AA and the 3′ adjacent 19 nucleotides. 3. G/C content < 50% is preferable. 4. BLAST search candidates, eliminating those with significant homology to other coding sequences. http://www.ambion.com/techlib/misc/siRNA_finder.html
Method #1: Custom siRNA Synthesis • Commercial synthesis of siRNA (~1-2 week turn around; getting quicker) • Expensive, but little to no hands-on time • Must screen siRNAs to identify an effective one • Synthesis can easily be scaled up • siRNAs can be labeled
Northern of GAPDH
Duration Results: mRNA Expression
Method #2: In vitro Transcription • In vitro transcribe sense and antisense RNA strands from dsDNA template (hybridized DNA oligonucleotides); hybridize RNA strands to create siRNAs, clean up • Inexpensive – a fraction of the cost of chemical synthesis • Fast turn around – synthesize and have ready for transfection in one day • Just as effective as chemically synthesized siRNAs, and can be used at lower concentration • Must screen siRNAs to identify an effective one • siRNAs can be labeled
• Obtain 2 desalted DNA oligonucleotides (with 8 bases complementary to T7 promoter primer)• Anneal oligonucleotides to T7 promoter primer• Fill-in reaction with Klenow• Transcribe with T7 RNA polymerase• Hybridize, digest and clean up
Method #3: RNase III/Dicer Digestion• Cocktail of several siRNAs generated by RNase III/Dicer digestion of long dsRNA• RNase III/Dicer Cocktails effectively induce RNAi in mammalian systems• RNase III/Dicer cleaves dsRNA into 12–30 bp dsRNA fragments with 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini.• No need to screen for effective siRNA• Cocktail of siRNAs provides better chance for strong RNAi effect on first try; typically no problems with nonspecific effects• Can label siRNA cocktail• Does not identify which siRNA sequence is effective
siRNA Cocktails Made with RNase III - Complementary RNA strands (100-500 nt) transcribed from dsDNA template and then hybridized to form long dsRNA. - DNase & RNase used to remove DNA template and unhybridized RNA strands. - RNase III digests dsRNA into population of 12-15mer dsRNAs that are functional as siRNAs. - Clean up reaction (removes long dsRNA) to ready it for transfection.
RNase III Generated siRNA Cocktails
Effect of GAPDH Cocktail on Other Genes
GENES EXAMINED
Method #4: siRNA Expression Vectors • Plasmids encoding siRNA sequences for expression in vivo by Pol III promoters (U6, H1) • Just as effective as chemically synthesized and in vitro transcribed siRNAs; same target sequences can be used • uses standard cloning techniques, eliminating need to synthesize or work with RNA • Can be used for transient expression or for transient selection and longer duration silencing when selectable markers are included • Long term gene silencing: - analysis of loss-of-function phenotypes within cell lines - potential for gene therapy
Example of Expression Vector Selectable Plasmids targeting GFP
Use of Plasmid Expression Vectors Step 1: Identify siRNA target sequence and design siRNA encoding DNA insert Step 2: Clone insert into siRNA Expression Vector Step 3: Select for clones containing insert, test, grow up Step 4: Transfect expression vector carrying insert into cells Step 5: Assay directly for RNAi after 18-48 hrs or select for transient expression or select for stable integration
Duration Studies: Long Term Reduction with Hygromycin Selectable Plasmid
Reduction of GFP after threeweeks of hygromycin selection
Method #5: siRNA Expression Cassettes (SECs) • Rapid, PCR-based method for preparing siRNA expression system • One-day turn around; avoids labor intensive cloning used with standard expression vector systems• Allows quick screening of siRNA target sequences and siRNA sequence : promoter combinations• SECs are easily inserted into receptor vectors (e.g. with selectable markers for long term duration studies)
• A precursor SEC generated by PCR using two gene specific DNA oligo nucleotides and primers provided in the kit (using a one-step or two-step PCR approach). The precursor SEC comprises an RNA pol III promoter and adjacent hairpin siRNA template.• The precursor SEC is used in a large scale PCR to generate the final SEC.• The SEC is column purified to remove primers, dNTPs, enzyme and salts to ensure efficient transfection
Transfecting Plasmids, SECS, siRNAs • Transfection is critical to success of RNAi expt; many variables affect efficiency • Efficiency of transfection agents can vary dramatically • Most are optimized for delivery of plasmid DNA, not PCR products or RNA; those optimized for RNA are for mRNA delivery and don’t perform well for siRNA • Key is to use agent optimized for plasmid, PCR or siRNA delivery – transfection efficiency must be high enough to be able to measure silencing
Plasmid & PCR Transfections • Protocols and reagents for plasmid transfection are numerous; efficiency is highly variable depending on cell line transfected (0-90%) • Conditions for plasmids are different than those for PCR products (SECs)
Cautions!!!!!!
Optimized siRNA Transfections
Two reagents optimized for siRNA delivery: • siPORT Amine - polyamine mixture • siPORT Lipid - mixture of cationic & neutral lipids
Silencer™ siRNA Transfection Kit • Contains two agents optimized for siRNA delivery • Includes well characterized GAPDH synthetic siRNA and a scrambled GAPDH negative control
GAPDH Scrambled
Labeled siRNAs: Applications • Analyze ability of siRNA to attenuate target gene expression • Determine transfection efficiency • Track siRNA migration within a cell • Study siRNA metabolism • Study siRNA in living cells in real time
siRNA = RedNucleus = BlueProtein = Green
GAPDH siRNA in HeLa S3 Cells
TetO mutTBP
TetR
Tet
TetR : Tet repressor TetO : Tet operator
mutTBP : mutant TBP wtTBP : wild type TBP
mutTATA box RNAi
mutTBPwtTBP
Human/mouse U6 promoter
pTRE/mutTBP
RNAi vector
Establishment of strategies for the conditional expression of shRNA
Tet-on inducible RNAi expression
RNAiTOH1 promoter
TetR
Tet
EMBO Rep. 2003 Jun;4(6):609-15.
Genetic Network for the Maintenance of Pluripotency in ES Cells; Application of pSTAT3ER/4-HT system
pSTAT3ER
+ LIF
pRNAi
+ 4-HT+/- LIF
- LIF- 4-HT
Synchronized differentiation
ESC ESC/pSTAT3ER ESC/pSTAT3ER/pRNAi
About siRNARNAi (RNA interference) is a phenomenon that small double-stranded RNA (Referred as small interference RNA or siRNA) can knock down the expression of its corresponding gene. RNAi has been observed in plant, C.elegans and Drosophila long time ago. It was until recently that RNAi was discovered to w
ork in mammalian system [1]. Small interference RNA (siRNA) is 19-22 nt double-stranded RNA. It works by cleaving and destroying its cognate RNA. siRNA first assembles into RNA-induced silencing complexes (RISCs), and it then activates the complex by unwinding its RNA strands. The unwound RNA strands subsequently guide the complex to the complementary RNA molecules, where the complex cleaves and destroys the cognate R
NA, which results in RNAi phenomenon.
RNAi has evolved into a powerful tool to study gene functions. Here are some of its applications: 1. A stable cell line with a specific gene knocked-out can be established, and its phenotype can be studied. 2. A knock-out mouse line can be established using trangenic siRNA method [8]. 3. siRNA can be put into a vector with an inducible promoter to study its effect. 4. siRNA can be delivered by using viral vector [6,7] and used for gene therapy purpose. 5. siRNA can be mimicked by chemical molecule and used for drug development.
DNA vector-based siRNA technologysiRNA can be obtained by chemical synthesis or by DNA-vector based RNAi technology.
Using DNA vector based siRNA technology, a small DNA insert (about 70 bp) encoding a short hairpin RNA targeting the gene of interest is cloned into a commercially available vector. The insert-containing vector can be transfected into the cell, and it expresses the short hairpin RNA. The hairpin RNA is rapidly processed by the cellular machinery into 19-22 nt double stranded RNA (siRNA).
The following is a list of GenScript siRNA expression vectors: 1. U6 like promoter: pRNA-U6.1/Neo pRNA-U6.1/Hygro H1 like promoter pRNA-H1.1/Neo pRNA-H1.1/Hygro
Advantages of DNA vector-based siRNA technologyComparing to chemically synthesized siRNA, DNA-vector based technology has a lot of advantages: 1.Vector based siRNA is more effective than synthetic siRNA for inhibition of gene expression [2]. 2.Very stable and easy to handle: Synthetic siRNA is not stable, which has to be protected during shipping and de-protected before use. Unlike synthetic siRNA, vector based siRNA is the same as DNA, and it is very stable and can be easily tranfected into cell using routine DNA transfection reagents, such as Lipofectamine. 3.Stable cell line can be established: vector based siRNA allows you to obtain a stable cell line, and observe long-term effects of RNAi. 4.Inducible system can be established: vector based siRNA allows you to establish an inducible system by using a vector with an inducible promoter. 5.A knock-out mouse line can be established using trangenic siRNA method [8]. 6.Unlimited supply: once a DNA construct is made, you will have unlimited supply of siRNA. 7.Cost-effective: synthetic siRNA has to be re-ordered once it is used up whereas vector-based siRNA only need to be ordered one time. One big obstacle for vector-based siRNA technology is that it takes a lot of time and trouble to make the DNA constructs. GenScript can provide you custom siRNA construct! Follow this link to Learn more about how Genscript siRNA technology works for you.
siRNA Target Site SelectionWe have developed some software tools to facilitate your design process. It is recommended that at least 3 vector-based siRNA should be prepared for each gene in order to find a potent and specific siRNA. Here are the reasons: 1.Not all siRNA target sequences are equally potent: Because of secondary structure and other factors, some target sequences are more potent than others. It is better to test at least three vector-siRNA constructs to find the most potent one. 2.Not all siRNA silencing effects are gene-specific: It has been reported that some siRNA silencing effects are not gene-specific because of various of reasons. It is better to validate your experiments results using three vector-based siRNA constructs. 3.Results from synthetic siRNA or siRNA cassette cannot be completely transferred to vector-based siRNA construct: Vector-based siRNA is different from synthetic siRNA oligos or siRNA cassette. Although the results from synthetic siRNA oligos or siRNA cassette can suggest the most potent siRNA targets, the results cannot be completely duplicated in vector-based siRNA for unknown reasons. 4.The experiment is still the gold test stone: Although we are pride of our vector-based siRNA design program, the best design is still not as good as what the experiments can tell you.
References 1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA i
nterference in cultured mammalian cells. Nature 411: 494-498. 2. Yu JY, DeRuiter SL, Turner DL. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mamma
lian cells. Proc Natl Acad Sci U S A. 99(9):6047-6052. 3. Brummelkamp, T.R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mamma
lian cells. Science 296: 550-553. 4. Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA interference. Nature 418: 435-
438. 5. Sui, G., Soohoo, C., Affar, E.B., Gay, F., Shi, Y., Forrester, W.C., and Shi, Y. (2002) A DNA vector-based RNAi technology t
o suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(8): 5515-5520. 6. Shen C, Buck AK, Liu X, Winkler M, and Reske SN. (2003) Gene silencing by adenovirus-delivered siRNA. FEBS Lett 539(1-
3):111-114. 7. Barton GM, and Medzhitov R. (2002) Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A
99(23):14943-14945. 8.Kunach T, Gish G, Lickert H, Jones N, Pawson T, and Rossant J. (2003) Transgenic RNA interference in ES cell-derived emb
ryos recapitulates a genetic null phenotype. Nature Biotechnology 21:559-561.