Inducing RNAi in Mammalian Cells

Transcription

1 Inducing RNAi in Mammalian Cells Ambion, Inc. sirna localized to the perinuclear space View at: Important Applications of RNAi Ascertain function of genes Create loss-of-function phenotypes Potential for therapeutics (e.g. HIV, Hepatitis C)

2 Biochemical Mechanism of RNAi dsrna is introduced into the cell. DICER digests dsrna into ~21bp ds RNA (short interfering RNAs; sirnas). The sirnas are integrated into the RNA Induced Silencing Complex (RISC). The sirnas undergo strand separation. The antisense strand then binds to its complementary/target mrna. Nucleases within the RISC degrade the targeted mrna. sirna Features and Synthesis Characteristics of sirna ds RNA of ~21 bp with 3 dinucleotide overhangs hairpins with bp stems and 4-9 nt loop sirnas can be produced by: Chemical synthesis Enzymatic synthesis RNase III/Dicer cleavage of long dsrna Plasmid based in vivo expression

3 sirna Design 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. (Provides tool for finding sirnas in mrna sequence) Parameters of sirna Effectiveness No correlation with target location Strong correlation with low G/C content Gene Location mrna G/C 5 75% 53% c-myc 5 Medial 83% 52% 3 Medial 11% 52% 3 6% 33% 5 82% 52% La 5 Medial 32% 33% 3 Medial 60% 43% 3 63% 43% 5 25% 38% GAPDH 5 Medial 7% 38% 3 Medial 36% 57% 3 45% 57%

4 Duration Results: mrna Expression Northern of GAPDH 28S rrna GAPDH H 24 H 3 Day 6 Day 10 Day 12 Day NT 4H 24H 3 Day 6 Day 10 Day 12 Day NT Custom sirna Synthesis Commercial synthesis of sirna (2+ weeks turn around) 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

5 Ambion Custom sirna Synthesis Licensed manufacturer of sirna oligonucleotides High quality synthetic sirnas Synthesized at 0.2 µmol scale; sufficient for s-0s of transfections dtdt overhangs are added to 3 ends as default; other nt overhangs can be specified Optimized deprotection and reverse phase desalting PAGE purification In vitro Transcription of sirnas 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

6 Transcription Method Transcription Method 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

7 Silencer sirna Construction Kit Lower cost sirnas Make up to 15 sirnas in less than 24 hrs Hundreds of transfections per reaction Control templates to generate GAPDH sirna included Synthetic vs In Vitro Transcribed sirnas

8 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 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 (-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.

9 RNase III Generated sirna Cocktails La Effects of 50nM RNAseIII Generated sirnas Effects of 50 nm sirnas Cocktails DAPI c-fos Protein % of Non-Transfected NT GAPDH GAPDH 14 NT La La NT c-fos c-fos 25 NT c-myc c-myc 35 Gene of Interest DAPI Protein RNase III Digestion Product Size Blue: DAPI Green: GAPDH NT bp region 21bp chemically synthesized RNase RNAse III Digested III Digested GAPDH GAPDH Cocktail - effect of Product different Size sized Comparison digestion products - % of Non-Transfected NT 12-15mer Chemically Synthesized 21mer Product Regions

10 sirna Cocktails Nonspecific Effects? Non-Specific Effects of RNAse III Digested GAPDH Effect of GAPDH Cocktail on Other Genes % of Non-Transfected Fluorescent Signal / Cell NT-GAPDH GAPDH La Ku-70 c-myc beta-actin cdk2 Genes Examined sirna Expression Vectors Plasmids encoding sirna sequences for expression in vivo by Pol III promoters (U6, H1) Can be used for transient expression, or for 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

11 sirna Plasmid Expression Vectors Can do transient transfection, transient selection or long term duration experiments Eliminates the need to synthesize or work with RNA Just as effective as chemically synthesized and in vitro transcribed sirnas; same target sequences can be used Must screen sirnas to identify an effective target site Construction is labor intensive sirnas cannot be labeled psilencer TM sirna Expression Vectors

12 Example of Expression Vector expression Use of Plasmid Expression Vectors Identify sirna target sequence and design sirna encoding DNA insert Clone insert into sirna Expression Vector Select for clones containing insert, test, grow up Transfect expression vector carrying insert into cells Assay directly for RNAi after hrs or Select for transient expression or Select for stable integration

13 RNAi Using psilencer TM 2.0-U6 & 3.0-H1 Cells transfected with psilencer 2.0-U6-GAPDH Transfected Nontransfected Duration Studies Long Term Reduction with Hygromycin Selectable Plasmid Reduction of GFP after 3 weeks using Hygromycin Selectable Plasmids Scr GFP % of Non-Transfected Fluorescent Signal GFP Empty Vector 4 U6 driven GFP sirna Plasmids

14 Transient Selection Studies Selectable Plasmids targeting GFP Scrambled Hygro Neo Puro U6 Promoter H1 Promoter H1 Promoter Transfecting sirnas Transfection critical to success of RNAi expt; many variables affect efficiency Efficiency of transfection agents can vary dramatically Most are optimized for delivery of DNA (plasmid or oligonucleotides); those optimized for RNA are for mrna delivery and don t perform well for sirna Key is to use agent optimized for sirna delivery

15 Optimized sirna Transfections Two reagents optimized for sirna delivery: siport Amine - polyamine mixture siport Lipid - mixture of cationic and 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 Silencer Transfection Results siport TM Lipid siport TM Amine GAPDH Scrambled

16 Cell Line Compatibility Cell Line HeLa/ HeLa S3 COS-7 CHO-K1 MCF-7 NIH/3T3 HT-29 A BJ Mammalian Cell Type Human cervical carcinoma Monkey kidney SV40- transformed Hamster ovary Human breast carcinoma Mouse embryo fibroblast Human colon adenocarcinoma Human lung carcinoma Human kidney transformed Human foreskin fibroblast Recommended Reagent siport Amine siport Lipid 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

17 Silencer sirna Labeling Kit Available for labeling with either Cy3 or FAM. Does not affect transfection efficiency or biological function. Can be used to label chemically synthesized, in vitro transcribed, and RNase III generated sirnas. Step 1 Step 2 Step 3 Effect of Label on sirna Activity 120% Relative protein level immunofluorescence No label Sense strand label Antisense strand label Double label NT % 80% 60% 40% 20% 0% c-myc sirna Scrambled sirna

18 Effect of Labeled sirna on Target Gene HeLa cells sirna to β-actin Non-Transfected With Cy3 Label Without Label sirna = Red Nucleus = Blue Protein = Green Analyzing Target Gene Reduction GAPDH sirna in HeLa S3 Cells GAPDH sirna = Red Nucleus = Blue Protein = Green Scrambled

19 sirna Localization What we have visualized: The sirnas localize to the perinuclear space Red: ds β-actin sirna (Cy3) Green: β-actin Ab (Fluorescein) Blue: nucleus (DAPI) Inducing RNAi in Mammalian Cells

20 Ambion s sirna Tools and Products RNAi Resource: sirna Chemical vs. In Vitro Synthesis Custom sirna Synthesis Silencer sirna Construction Kit RNase III; Silencer sirna Cocktail Kit Expression Vectors psilencer Vectors: - 3 promoters - 3 selectable markers Ambion s sirna Tools and Products Optimizing Transfection of sirnas Silencer sirna Transfection Kit siport Amine siport Lipid Uses of Labeled sirna Silencer sirna Labeling Kit

21 Acknowledgements Research & Development Ambion, Inc. David Brown, Ph.D. Lance Ford, Ph.D. Rich Jarvis Mike Byrom Vince Pallotta Tu Khoeunh Anton Zimmerman Ann Ott Angie Cheng