1. Genome Scale Reagents and Services for Functional Studies January 21st - 22nd, 2014 Biomedicum Functional Genomics Unit (FuGU) RECOMBINANT VIRUS PRODUCTS AND qRT-PCR SERVICES AVAILABLE AT FuGU

2. Established in 2006, operates under the national Biocenter Finland infrastructure network Co-directed by Outi Monni (genome profiling) and Juha Klefström (recombinant virus technologies) Located at Meilahti campus of the University of Helsinki –Biomedicum Helsinki 1 building Services include e.g. next-generation sequencing, microarrays by four commercial platforms, recombinant virus services and genome-scale reagents for gene knockdown January 21st-22nd, 2014FuGU2 Biomedicum Functional Genomics Unit

3. Recombinant Virus Products and Services Available at FuGU

4. Genome scale shRNA library for RNA interference -mediated gene silencing Both human and mouse TRC1 shRNA libraries available –developed by the RNAi Consortium at the Broad Institute of MIT and Harvard –~159,000 pre-cloned shRNA constructs targeting ~16,000 annotated human and ~15,950 mouse genes - in pLKO.1 lentiviral vector backbone on average, 3-5 constructs per gene Two in five clones will typically provide at least 70 % knockdown TRC1 shRNA library controls: –Empty pLKO.1 vector –Non-target (scramble) shRNA in pLKO.1 backbone –TurboGFP in pLKO.1 backbone –eGFP shRNA in pLKO.1 backbone same controls for human and mouse libraries January 21st-22nd, 2014FuGU4 The RNAi Consortium (TRC) shRNA library TRC Library Database: http://www.broadinstitute.org/rnai/public/ *shRNA (short hairpin RNA): Mimics miRNA by forming a hairpin structure, utilizes the endogenous RNAi pathway both in the nucleus and in the cytosol. Can be integrated into the host genome using viral vectors, transcribed by RNA polymerase III.

5. January 21st-22nd, 2014FuGU5 TRC1 pLKO.1 shRNA transfer vector Non-target shRNA control plasmid. Modified from: http://www.sigmaaldrich.com/life-science/functional-genomics- and-rnai/shrna/library-information/vector-map.html Wide variety of shRNA transfer vectors available Vector usually includes: RNA Pol III promoter (usually U6 or H1 shRNA insert) regulatory factors promoters for transcription in packaging cells antibiotics for bacteria carrying the plasmid Additional components: Selection marker (e.g. antibiotic resistance) Fluorescent reporter (e.g. GFP) Transgene shRNA

6. TRC1 library shRNA consructs are available from FuGU:  bacterial glycerol stocks  transfection grade plasmid DNA  Lentiviral particles for recombinant virus transduction (=infection) January 21st-22nd, 2014FuGU6 The RNAi Consortium (TRC) shRNA library

7. Viral mediated gene (DNA) transfer Lentiviral Particles –VSV-G pseudotyped (broad range of mammalian ja non-mammalian host cells) –MINI scale: 1.5 ml virus particles per construct –MIDI scale: 6 ml virus particles per construct –Concentrated Viral Particles: 180 ul / 380 ul, ~ 100-fold concentrated + Quality controls: Capsid titer test (p24), internal control TRC1 shRNA library constructs Customers own lentiviral (e.g. shRNA, shRNAmir or cDNA) vectors, e.g. from GBU Retroviral Particles –Ecotropic (mouse and rat) and Amphotropic (broader host range) retroviruses available –Moloney mouse leukemia virus (MMLV) and mouse stem cell virus (MSCV) based –MINI scale: 1.5 ml virus particles per construct –MIDI scale: 6 ml virus particles per construct Customers own retroviral vectors January 21st-22nd, 2014FuGU7 Recombinant Viral Particles

8. January 21st-22nd, 2014FuGU8 Viral Particle Production & Transduction 1.1 Lentiviral particles are created by co-transfecting packaging vectors + gene transfer vector into packaging cells 1.2 Retroviral particles are produced by packaging cells stably transcribing retroviral packaging plasmids 2. Viral particles are harvested from the cell culture supernatant 3. Target cells are transduced

9. Reasons to concentrate viral particles: –virus p24 titer is usually 10 4 -10 5 pg/ml (~10 6 infectious units / ml with HeLa cells) –particular cell lines require a high viral particle concentration to be infected (e.g. primary cells) –infections need to be done in a small volume –in vivo infection volumes are extremely small, whereas the viral particle concentration has to be very high At FuGU, lentiviral particles are concentrated utilizing ultracentrifugation –typically a 100-fold concentration is achieved Concentrated Lentiviral Particles 9January 21st-22nd, 2014FuGU

10. Reasons to purify lentiviral particles –Although sterile filtered, the viral particles may contain remnants of cells and media  possible inflammatory reaction when the viral particles are used in in vivo applications Sucrose Purification of Lentiviral Particles 10January 21st-22nd, 2014FuGU

11. Virus titer is analyzed to evaluate the quality of the recombinant virus product Two complementary methods: –p24 capsid titer test –Live virus titer analysis Most of FuGU’s lentiviral products are analyzed with the p24 test Lentivirus titer testing 11January 21st-22nd, 2014FuGU

12. p24 Capsid Titer Test –p24 (MW 24 kDa) is a major structural component of the viral capsid in recombinant lentiviruses –p24 concentration (pg/ml) is measured from the cell culture supernatant by a p24-specific ELISA assay –the test measures both functional viral particles and free p24 in the supernantant  the test alone does not give an exact amount of infectious particles Live Virus Titer Analysis –constructs containing a fluorescent protein coding sequence (e.g. GFP) –cells are transduced, the percentage of fluorescent-positive (i.e. infected) cells is calculated and compared to the amount of cells and the volume of viral supernatant used  analyzes the number of infectious units in the sample (IU/ml) Lentiviral Particles - Virus titer testing 12January 21st-22nd, 2014FuGU

13. RCV test should always be done before transduced, live cells are moved from BSL II to BSL I facilities ! Recombinant viruses are replication incompetent, but recombination enabling virus replication is theoretically possible Not all viral particles used in infection are transported into cells; the rest stay infectious until removed from the media (e.g. splitting of the cells) –Biohazard to people –Contamination risk for non-infected cell lines Replication competent lentivirus and viral particles can be detected from the infected cells’ media by measuring the concentration of p24 capsid protein –Samples with a  p24  below a certain threshold are regarded as RCV- negative RCV (Replication Competent Virus) testing 13January 21st-22nd, 2014FuGU

14. Managed Biosafety level II laboratory for recombinant virus work for registered customer –Biomedicum I, 5 th floor Provides biosafety training and registration Viral titer (p24 test) and RCV testing Possibility to purchase viral gene transfer products: –packaging cells for lenti- and retroviral particle production –other common cell lines relevant to virus work –transfection reagents Materials and reagents found at the BSL II facilities are included in the hourly fee Biomedicum Virus Core (BVC) 14January 21st-22nd, 2014FuGU

15. In qRT-PCR the cDNA amplification can be detected and quantified in real time at each PCR cycle, giving a quantitative measurement of the cDNA accumulation during the PCR Quantitative Real-Time PCR (qRT-PCR) services are offered for: –knockdown validation in human or mouse (e.g. TRC1 shRNAs) –validation of ectopic expression (Hs or Mm) –gene expression studies of up to 10 transcripts LightCycler ® 480 Instrument II and Universal ProbeLibrary (UPL) probes (Roche) Service includes: –RNA extraction (on request) –RNA quality control analysis (Bioanalyzer) –primer-probe design –cDNA synthesis –qPCR and data analysis January 21st-22nd, 2014FuGU15 qRT-PCR Services

16. UPL probes are hydrolysis probes similar to TaqMan® –only 8-9 nt long & optimized sequences  anneal to multiple sites of the transcriptome  specificity is achieved by combining a probe with target-specific primers –contain locked nucleic acids (LNA technology), increased specificity –labeled at the 5' end with fluorescein (FAM), at the 3' end with a dark quencher dye 90 UPL probes to cover the whole human, mouse and rat transcriptome –additional 75 probes for multiple organisms Enables multiplex assays: GOI and reference gene –10 reference gene assays for human, 2 for mouse Probes and primers for any assay can be found from Roche’s Universal ProbeLibrary Assay Design Center January 21st-22nd, 2014FuGU16 qRT-PCR - Universal ProbeLibrary (Roche)

17. January 21st-22nd, 2014FuGU17 Summary - Products and Services Available TRC1 shRNA library constructs shRNA plasmid DNA purification Transfections Lentiviral particles Virus titer testing Target cell transduction Selection of infected cells RNA extraction qRT-PCR knockdown validation Data analysis Results: knockdown efficiency FuGU’s Service Custome r provided e.g. knockdown validation service Step 1 Step 2Step 3Step 4 Step 5 Step 6 Step 7Step 8 Step 9 Step 10

18. Public TRC Portal: http://www.broadinstitute.org/rnai/public/ Sigma, MISSION RNAi: http://www.sigmaaldrich.com/life-science/functional- genomics-and-rnai/shrna/library-information/vector-map.html FuGU: http://www.helsinki.fi/fugu/index.html Addgene: http://www.addgene.org/lentiviral/packaging/ jetPEI™ transfection protocol: http://www.polyplus-transfection.com Universal ProbeLibrary System Technology, Roche: http://www.roche- applied-science.com January 21st-22nd, 2014FuGU18 References

19. January 21st-22nd, 2014FuGU19 Biomedicum Functional Genomics Unit Contact Information: Email: fugu-support@helsinki.fi Phone: Recombinant Virus Services: +358 9 191 25494 Genome Profiling Services: +358 50 442 0228 Web pages: www.helsinki.fi/fugu Personnel Outi Monni, PhD Juha Klefström, PhD Taru Perttula, B.Eng Hanna Pesonen, Lab Technician Maria Lehtivaara, M.Sc. (Tech.) Pauliina Munne, PhD Hanna Ala-Hongisto, M.Sc. Tiina Neejärvi, Laboratory Analyst Topi Tervonen, PhD Minna Lähteenmäki, M.Sc. (Tech.)