RNA Interference

1. RNA Interference Iain Fraser

2. Initiation step (RNA-induced silencing complex) Dicer Effector step Model for RNAi mechanism Hammond et al., 2001

3. U6 Expression Cassette H1 Expression Cassette +1 +1 U6 pr. G-N18-TT-loop-N’18-C TTTT H1 pr. N19-TT-loop-N’19 TTTT Pol III Pol III N19 N19 UU UU 5’- 5’- Short hairpin RNA N’19 N’19 UU UU Processing Processing N19 N19 UU UU 5’- 5’- siRNA duplex -5’ -5’ N’19 N’19 UU UU Expression of siRNAs from pol III promoters

4. Target sequence selection • Length: 19 basepairs • Original Tuschl rule of selecting N19 immediately following a AA is not necessarily observed in our design • %GC content: 45-55%, optimum 50% • Tm: 45-65oC, optimum 55oC • Avoid AA at start and TT at end of sequence to prevent premature termination • BLAST to ensure specificity • We find selection of an effective target sequence to be arbitrary

5. 4 linkers designed per gene ccdB counter-selection prevents background in cloning step Hairpin Linker + pEN_mH1c BamH1 Xho1 attL1 mH1 ccdB attL2 YFP Gene attL1 mH1 attL2 Co-express hairpins with YFP tagged gene of interest to identify effective hairpin Vector design

6. 188 98 Zeo control PTEN -C PTEN -D PTEN -A PTEN -B 62 49 38 28 17 14 Is data from transient expression reliable? • Best hairpin against heterologously expressed gene is also most effective against endogenous gene after selection of transfected cells Hairpins Control A B C D YFP-PTEN PTEN Transient transfection Zeocin selection

7. RNAi target genes

8. RNAi target genes

9. CXCR5-YFP YFP-Syk YFP-Gi2 M A B C D M X A B C D Hairpins: M A B C D IB: anti-YFP Ab Efficacy of CXCR5, syk and Galpha i2 hairpins • Hairpins and YFP tagged gene were used to co-transfect P19 cells • Cells were harvested 48hr post-transfection • Mock control was vector containing hairpin against different gene • X control in Galpha i2 experiment was chemically synthesized siRNA duplex directed against Galpha i2

10. Hairpin pEN_mH1c attL1 mH1 attL2 + pDSL_UGIP LTR FLAP attR1 attR2 Ubi-C GFP IRES Puro WRE LTR LR site specific recombination reaction pL_hp-UGIP LTR FLAP attB1 attB2 Ubi-C GFP IRES Puro WRE LTR mH1 Hairpin Subcloning of siRNA cassettes into lentiviral constructs

11. Infection of WEHI231 cells with siRNA-CXCR5 and siRNA-syk lentiviruses

12. CXCR5 siRNA stable Syk siRNA stable CXCR5 vector Syk vector Genomic PCR confirms integration of pol III cassettes • PCR carried out with sense primer in H1 promoter upstream of hairpin and antisense primer in ubiquitin promoter downstream of hairpin • PCR products excised from gel and sequenced • Sequence confirms both Syk and CXCR5 hairpins integrated in stable lines

13. CXCR5 FACS Data Syk Western Blot Integrated siRNAs have no effect on target protein in transduced WEHI cells

14. Integrated siRNAs have no effect on target mRNA in transduced WEHI cells Quantitative PCR Microarray

15. Gi2 primer Gi2 siRNA Gi3 siRNA Gi1 siRNA Control 1.2 1.0 0.8 Blot: anti-Gi2 Expression of mRNA(normalized with GAPDH) 0.6 0.4 Blot: anti-tubulin 0.2 0.0 Gi3 Control Gi2 SiRNA Targeting of G alpha i2 in J774A.1 Monocytes • Cells infected with lentivirus containing Galpha i2-C hairpin • Same UGIP lentiviral backbone as used for Syk and CXCR5 viruses • Data from puromicin selected cells mRNA Protein Jong-Ik Hwang, Simon Lab

16. GFP-Luc lacZ si-lacZ CMV CMV pol III Target cell Reporter assay for assessment of RNAi capacity • Assay for lacZ and luciferase activity 48hr post-transfection • Assay is very sensitive, giving data from <5% transfection efficiency

17. RNAi reporter assay: WEHI231 Beta-Gal Activity, RU +- ++ ++- ++ LacZ siLacZ ++++

18. RAW 264.7 B-Gal Activity, RU +- + + + ++ + +++ + ++++ LacZ siLacZ J774A.1 B-Gal Activity, RU +- ++ + ++ LacZ siLacZ RNAi reporter assay: J774A.1 and RAW264.7 • Transfection efficiency of J774s very low • Required electroporation to achieve detectable lacZ activity • LacZ activity at lower limit of assay sensitivity

19. 3T3L1 IC-21 B-Gal Activity, RU B-Gal Activity, RU +- ++ + ++ LacZ siLacZ +- ++ + ++ LacZ siLacZ N1E-115 B-Gal Activity, RU +- ++ + ++ LacZ siLacZ RNAi reporter assay: 3T3L1, IC-21 and N1E-115

20. Lentiviral-mediated RNAi in RAW264.7 cells:1 • Three siRNA hairpins designed against TREM-2B receptor • siRNA-expressing lentivirus used to transduce RAW264.7 cell line containing stably expressed FLAG-TREM-2B • siRNA efficacy assessed by FACS detection of FLAG tag Tamara Roach, AfCS Assay Lab

21. Lentiviral-mediated RNAi in RAW264.7 cells: 2 Tamara Roach, AfCS Assay Lab

22. Summary/Conclusions • We have established a flexible vector-based system for the development of effective siRNAs against genes of interest to the AfCS. • Expression of such siRNAs from lentiviral vectors permits the transduction of hematopoietic cell lines. • WEHI231 cells transduced with siRNAs do not exhibit RNAi-mediated target knockdown. It remains unclear whether this is due to the siRNAs not being expressed from pol III promoters, or the absence of the components of the RNAi machinery required to recognize the siRNAs. • Vector-based RNAi was effective in six other cell lines tested by reporter assay: J774A.1, RAW264.7, IC-21, N1E-115, 3T3-L1 and HEK293. • Lentiviral-mediated RNAi was effective in both J774A.1 and RAW264.7 cells

23. Acknowledgements Molecular Biology Lab Joelle Zavzavadjian Pamela Eversole-Cire Jamie Liu Dan Allen Mei Wang Sangdun Choi Assay Lab Tamara Roach Caltech Jong-Ik Hwang Melvin Simon