Transcript Functional Genomics Unit (FuGU)

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
Biomedicum Functional Genomics Unit
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Established in 2006, operates under
the national Biocenter Finland
infrastructure network
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Co-directed by Outi Monni (genome
profiling) and Juha Klefström
(recombinant virus technologies)
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Located at Meilahti campus of the
University of Helsinki
– Biomedicum Helsinki 1 building
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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, 2014
FuGU
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Recombinant Virus Products and
Services Available at FuGU
The RNAi Consortium (TRC) shRNA library
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Genome scale shRNA library for RNA interference -mediated gene
silencing
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Both human and mouse TRC1 shRNA libraries available
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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
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on average, 3-5 constructs per gene
Two in five clones will typically provide at least 70 % knockdown
TRC1 shRNA library controls:
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Empty pLKO.1 vector
Non-target (scramble) shRNA in pLKO.1 backbone
TurboGFP in pLKO.1 backbone
eGFP shRNA in pLKO.1 backbone
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same controls for human and mouse libraries
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.
January 21st-22nd, 2014
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TRC1 pLKO.1 shRNA transfer vector
shRNA
• Wide variety of shRNA transfer vectors
available
• Vector usually includes:
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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:
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Non-target shRNA control plasmid. Modified from:
http://www.sigmaaldrich.com/life-science/functional-genomicsand-rnai/shrna/library-information/vector-map.html
January 21st-22nd, 2014
FuGU
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Selection marker (e.g. antibiotic
resistance)
Fluorescent reporter (e.g. GFP)
Transgene
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The RNAi Consortium (TRC) shRNA library
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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, 2014
FuGU
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Recombinant Viral Particles
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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
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Retroviral Particles
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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
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Customers own retroviral vectors
January 21st-22nd, 2014
FuGU
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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
January 21st-22nd, 2014
FuGU
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Concentrated Lentiviral Particles
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Reasons to concentrate viral particles:
– virus p24 titer is usually 104-105 pg/ml (~106 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
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At FuGU, lentiviral particles are concentrated utilizing
ultracentrifugation
– typically a 100-fold concentration is achieved
January 21st-22nd, 2014
FuGU
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Sucrose Purification of Lentiviral Particles
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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
January 21st-22nd, 2014
FuGU
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Lentivirus titer testing
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Virus titer is analyzed to evaluate the
quality of the recombinant virus product
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Two complementary methods:
– p24 capsid titer test
– Live virus titer analysis
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Most of FuGU’s lentiviral products are
analyzed with the p24 test
January 21st-22nd, 2014
FuGU
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Lentiviral Particles - Virus titer testing
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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
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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)
January 21st-22nd, 2014
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RCV (Replication Competent Virus) testing
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RCV test should always be done before transduced, live cells
are moved from BSL II to BSL I facilities!
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Recombinant viruses are replication incompetent, but
recombination enabling virus replication is theoretically possible
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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
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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 RCVnegative
January 21st-22nd, 2014
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Biomedicum Virus Core (BVC)
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Managed Biosafety level II laboratory for recombinant virus work for
registered customer
– Biomedicum I, 5th floor
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Provides biosafety training and registration
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Viral titer (p24 test) and RCV testing
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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
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Materials and reagents found at the BSL II facilities are included in
the hourly fee
January 21st-22nd, 2014
FuGU
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qRT-PCR Services
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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
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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
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LightCycler® 480 Instrument II and Universal ProbeLibrary (UPL)
probes (Roche)
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Service includes:
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RNA extraction (on request)
RNA quality control analysis (Bioanalyzer)
primer-probe design
cDNA synthesis
qPCR and data analysis
January 21st-22nd, 2014
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qRT-PCR - Universal ProbeLibrary (Roche)
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UPL probes are hydrolysis probes similar to TaqMan®
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90 UPL probes to cover the whole human, mouse and rat
transcriptome
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additional 75 probes for multiple organisms
Enables multiplex assays: GOI and reference gene
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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
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, 2014
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Summary - Products and Services Available
e.g. knockdown validation service
Step 1
TRC1 shRNA
library
constructs
Step 5
Target cell
transduction
FuGU’s
Service
Custome
r
provided
January 21st-22nd, 2014
shRNA
plasmid DNA
purification
Step 2
Transfections
Step 3
Lentiviral
particles
Step 4
Virus titer
testing
Step 6
Selection of
infected cells
Step 7
RNA
extraction
Step 8
qRT-PCR
knockdown
validation
FuGU
Step 9
Data
analysis
Step 10
Results:
knockdown
efficiency
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References
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Public TRC Portal: http://www.broadinstitute.org/rnai/public/
Sigma, MISSION RNAi: http://www.sigmaaldrich.com/life-science/functionalgenomics-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.rocheapplied-science.com
January 21st-22nd, 2014
FuGU
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Biomedicum Functional Genomics Unit
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.)
Contact Information:
Email: [email protected]
Phone:
Recombinant Virus Services: +358 9 191 25494
Genome Profiling Services: +358 50 442 0228
Web pages: www.helsinki.fi/fugu
January 21st-22nd, 2014
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