Databinge AAV Presentation
Matthieu Vanni + Alexa Nelson
Table of Contents
Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Addgene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Transfection and Transduction . . . . . . . . . . . . 5 - 9
AAVs General Info . . . . . . . . . . . . . . . . . . . . . 10 -12
AAV Serotypes . . . . . . . . . . . . . . . . . . . . . . . . 13 - 18
AAV Promoters . . . . . . . . . . . . . . . . . . . . . . . 19 - 36
Buying AAVs . . . . . . . . . . . . . . . . . . . . . . . . . . 37 - 38
AAV Biosafety . . . . . . . . . . . . . . . . . . . . . . . . 39 - 40
Handling AAVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Animal Care . . . . . . . . . . . . . . . . . . . . . . . . . . 42 - 43
Rat Stereotaxic Surgery for AAV Injection . 44 - 59
Animal Resource Unit + Follow-up . . . . . . . 60 - 62
Image Examples in Brain Slices . . . . . . . . . . 63 - 68
Image Examples in vivo . . . . . . . . . . . . . . . . 69 – 76
Additional Links . . . . . .. . . . . . . . . . . . . . . . . . . . . 77
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Plasmid DNA
• Plasmid = double-stranded DNA that can
replicate independently of chromosomal DNA
• Found in bacteria
• 1,000 – 20,000 base pairs (1-20 kbp)
• Used to code for probes like GCaMP
https://www.addgene.org/mol-bio-reference/plasmid-background/
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Addgene
• Non-profit plasmid repository
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https://www.addgene.org
Plasmids stored in bacteria in stabs (what is mailed)
Great resource for information about molecular biology, viruses, etc.
Downloadable Molecular Biology Reference
• Navigating the website:
1.
Search
2.
Sort + Filters to narrow down
3.
Select plasmid
4.
Literature Search
• Eg. mitochondrial calcium
• Eg. popularity + mammalian expression
• Eg. CMV-mito-R-GECO1 (#46021)
• Zhao Y, Araki S, Wu J, Teramoto T, Chang Y-F, Nakano M, et al (2013) An
expanded palette of genetically encoded Ca2+ indicators. Science 333: 18881891.
https://www.ncbi.nlm.nih.gov/pubmed/?term=21903779
https://www.addgene.org/recipient-instructions/streakplate/
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Transfect or Transduce Plasmid
1. Isolate plasmid DNA (pDNA) from Addgene bacterial stab
• Eg. with QIAGEN midiprep kit
• https://www.qiagen.com/us/?akamai-feo=off
2. Transfect DNA:
• Lipofection
• (eg. Lipofectamine [Invitrogen], Effectene [Qiagen], Lipid Nanoparticles)
• Electroporation
• Microinjection
• Biolistics
3. OR, transduce DNA using viruses:
• Package DNA in viruses
• Purchase pre-packaged from provider like Laval or UPenn
• Cheap
Plasmid from
Addgene
Isolate pDNA
Transfect
Transduce
(eg. in mammalian cultures, in vivo)
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Transfection
• Advantages:
• Simple + fast
• Less biosafety/biohazard paperwork
• Disadvantages:
• Can be less efficient (eg. with lipofection)
• Examples of reagents:
• Lipofectamine (Thermofisher)
http://www.thermofisher.com/ca/en/home/life-science/cellculture/transfection/transfection-reagent-application-table.html
• Lipid Nanoparticles (LNPs):
http://www.liposomes.ca
• Dr. Pieter Cullis @ UBC
• Packaging size limit?
Example of transfection protocol
(Thermo Fisher Scientific)
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Transduction with Viruses
• Advantages:
• Efficient
• Disadvantages
• More paperwork
• Packaging size limits
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Transduction
https://www.addgene.org/viral-vectors/
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Summary of Transduction and Transfection Methods in Neurons
• Karra D, Dahm R (2010) Transfection techniques for neuronal cells.
30(18): 6171-6177.
https://www.ncbi.nlm.nih.gov/pubmed/?term=20445041
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Adeno-Associated Viruses (AAVs)
• Wild-type AAV:
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Parvovirus not associated with pathology
~ 20 nm
ssDNA ~4.7 kb
Genome:
• 2 inverted terminal repeats (ITRs)
• 2 open reading frames (ORFs)
• Rep (4 proteins in replication) = Rep78, Rep68, Rep52, Rep40
• Cap (3 proteins in capsid formation) = VP1, VP2, VP3 (virion particle)
• Integrates into AAVS1 on human chromosome 19
• Good resources for information:
• https://www.addgene.org/viral-vectors/aav/aav-guide/
• http://ppn.med.sc.edu/Adeno-associated.Virus.asp
• http://vectorbiolabs.com/vbs/page.html?m=281
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AAVs in Research
• In research use recombinant AAV
(rAAV) depleted of rep and cap
genes
• Virus does not replicate
• Only leaves ITRs around gene of
interest
• Maintained extrachromosomally
http://ppn.med.sc.edu/Adeno-associated.Virus.asp
• Many serotypes
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Recombinant AAVs (rAAVs)
• Produce recombinant AAVs by cotransfecting 3 plasmids in cell line:
1. rAAV vector (gene of interest with ITRs)
2. AAV replication and cap genes
3. Helper genes
• Isolate virus
• Many companies do this, cheap to buy
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AAV Serotypes
• AAV2 one of most widely used serotypes but low transduction efficiency
• AAV Pseudotyping
• AAV2 genomes in capsids from other AAVs
• Eg. rAAV2/5
• Genome based on AAV2, capsid based on AAV5
• Tropism should be same as AAV5
• AAV2/5 more widely distributed in brain, better transduction efficiency than AAV2/2
• Multiple serotypes, eg. AAV-DJ hybrid capsid from eight serotypes
• Higher transduction efficiency compared to wt
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AAV Serotypes
UPenn
Addgene
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AAV Serotypes
Retrograde transport
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Wide expression
So which serotype to use?
1. Read the literature to see which serotype would be optimal
• Aschauer DF, Kreuz S, Rumpel S (2013) Analysis of transduction efficiency, tropism and axonal
transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. Plos One 8: 1-16.
https://www.ncbi.nlm.nih.gov/pubmed/?term=24086725
• Watakabe A, Ohtsuka M, Kinoshita M, Takaji M, Isa K, Mizukami H et al (2015) Comparitive
analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and
macaque cerebral cortex. Neuroscience Research 93: 144-157.
https://www.ncbi.nlm.nih.gov/pubmed/?term=25240284
2. Test for yourself what is optimal in your system
• GFP-expressing AAV2 serotypes from Harvard at low cost
• https://vectorcore.mgh.harvard.edu/AAV_guide
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Aschauer DF et al. (2013) Paper
Expression
Inflammatory Response
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AAV 2/9 for Brain
• AAV9 has good spread
• But depends on what the study question is
• eg. might not be appropriate if doing tracing
• AAV2/9 recommended by Harvard for brain
• Wade Regehr (Hosted by Dr. Ann Marie Craig)
• AAV2/9 with Synapsin promoter
• Jackman SL, Turecek J, Belinsky JE, Regehr WG (2016) The calcium sensor
synaptotagmin 7 is required for synaptic facilitation. Nature 529: 88-91.
https://www.ncbi.nlm.nih.gov/pubmed/?term=26738595
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AAV Grammar
Promoter
serotype
Sequences of interest
(will be transcripted/translated to the precious protein wanted)
“-” dash for fusion proteins
“.” dot to separate sequences
AAV1.Syn.flex.ChR2-GFP.WPRE.SV40
Extrinsic Specificity
(Optional)
AAV1.Syn.cre
Associated sequences
(won’t be expressed but will impact the
expression of the sequences of interest)
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Promoters (Mammalian Applications)
• General expression
• CMV: Cytomegalovirus
• CBA (emCBA: enhance mini): chicken β actin (CI: CBA intron?)
CaMK2a-tTA mouse
(jax 003010)
• CMV enhancer + CBA = CAG (or CB7) synthetic promoter, CAGGS(?)
• EF1a: Human elongation factor-1 alpha.
• PGK1: phosphoglycerate kinase
• Ubc: ubiquitin C
• Specific expression
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Syn (hSyn, hSyn1): synapsin promoter  every neurons
CaMKIIa  excitatory neurons
hThy1  excitatory neurons
GFAP (Glial fibrillary acidic protein)  glial cells
• GfaABC1D or GFA104 (0.7kb): compact GFAP (2.2kb)
TH: Tyrosine hydroxylase  dopamine cells
MCK: muscle creatine kinase  myocytes
cTNT: Chicken Cardiac Troponin T  cardiomyocytes
TBG: Thyroxine-binding globulin  hepatocytes
• Tetracycline dependant
• TRE: tetracycline transactivator (tTA) protein (Tet-ON/Tet-Off system)
https://www.addgene.org/tetracycline/
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Other Sequences
• WPRE: (Woodchuck Hepatitis Virus)
Posttranscriptional Regulatory
Element. Enhance the expression.
• poly A sequences (SV40, hGH, BGH,
rBG, NRN …): Motif AAUAAA which
promotes both polyadenylation and
termination. Define the end of a
transcription.
• e.g. AAV1.Syn.GCaMP6s.WPRE.SV40
• WGA (wheat germ agglutinin)
transported trans-synaptically used
as a circuitry tracer.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651700/
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Multicistronic Vectors
• IRES Elements
• poliovirus RNA, 600b!
• Multiple translation site
• 2A (x2A) sequence (e.g. P2A, GSG.P2A)
• foot-and-mouth disease virus, super short
• self-cleaving
• e.g. AAV1.Syn.GCaMP6s.P2A.NES-jrGECO1a
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Common gene of interest and fusion proteins/sequence
• Functional Neurophotonics and other genetic engineering
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Calcium sensors : GCaMP (6s, 6m, 6f, 3), jrGECO1a, jRCaMP1a, CaMPARI, Twitch2B
Glutamate sensor : iGluSnFR
Voltage sensor: ArcLight
Optogenetic inhibitor: Jaws, Arch, NpHR, …
Optogenetics activator: ChR2 (many), ChETA, Chronos, ChrimsonR, C1V1
Pharmacogenetics (DREADD): hM3D, hM4D
Diphtheria toxin A fragment: dtA
• Immunohistochemistry tag:
• HA: hemagluttinin (e.g. HA-hM4D)
• Trafficing
• NES, H2B, NLS: nuclear localization sequence (fusion protein, e.g. NES-jrGECO1a)
• Lck, ER2, KGC, TS, Myr: membrane localization (e.g. Lck-GCaMP6)
• Fluorescent fusion proteins:
GFP, YFP, (e.g. ChR2-GFP)
mCherry, tdTomato, dsRed, …
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Cre/Lox Recombination
Cre recombinase recombine LoxP sites (34b)
• Cre, creERT2 (Tamoxyfen inductible), cre-GFP
• e.g. AAV1.Syn.cre
Cre-dependant strategies:
• gene knock-out: “floxing”, for “flanked by loxP”
• LoxP – x – LoxP
• Cre-dependant gene expression: “lox-stop-lox” or “LSL”
cassette
• e.g. AAV1.CAG.LSL.tdTomato.bGH
• flex (“flip-excision”) or DIO (Doublefloxed Inverse Orientatio) or or double flox (dflox)
• flex-frwd: cre  inactivation by inversion
• e.g. AAV1.Syn.Flex.GCaMP6f.WPRE.SV40
CAG
stop
LSL:
CAG
tdTomato
tdTomato
stop
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Cre/Lox Alternative and Association
• Flippase Flp vs. FRT site
• e.g. AAV-Syn-FLPo
• Dre recombinase vs. rox site
• Combining the cre/lox, flp/frt and dre/rox: INTRSECT (INTronic
Recombinase Sites Enabling Combinatorial Targeting, Fenno et al.
2014 Nature Methods): Bolean logic
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e.g.
AAV-nEF-Con/Fon hChR2(H134R)-EYFP-WPRE
AAV-nEF-Con/Foff hChR2(H134R)-EYFP-WPRE
AAV-nEF-Coff/Fon hChR2(H134R)-EYFP-WPRE
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Where to buy AAVs?
• Canadian Neurophotonic Plateform (Univ. Laval):
http://neurophotonics.ca/molecular-tools-platform
• Upenn (Univ. of Pennsylvania):
http://www.med.upenn.edu/gtp/vectorcore/
• Vector Biolabs http://www.vectorbiolabs.com/Recombinant-AAV/ ?
• UNC (Univ. of North Carolina):
http://www.med.unc.edu/genetherapy/vectorcore
Never tried:
• Stanford: http://med.stanford.edu/gvvc/stockviruses.html
• Duke: https://sites.duke.edu/dvvc/collection-aav/
• Addgene (Cambridge, MA): http://www.addgene.org/viralservice/aav-prep/
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Serotypes mostly 1, 5, 9
Synapsin promoter
CaMPARI: Switchable GCaMP
WPRE: enhancer
polyA sequence = stop
Different GCaMP
CAG promoter
CaMK2 promoter
NES: nuclear localization
jrGECO1a: red GCaMP6
jRCaMP1a: red GCaMP6
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iGluSnFR: glutamate sensor
GFAP promoter
Twitch2B: FRET calcium indicator
mRuby2: red reporter
GfaABC1D GFAP promoter
2A: self-cleaving
Lck=membrane localization
Jaws-KGC: red activated chloride pump targeted to
membrane
hGH,bGH polyA
tdTomato, GFP fusion protein
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EF1a promoter
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Flex inversion
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gene knock-out: “floxing”
2A cleavage
CBA promoter
DIO=flex
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Cre express under CMV promoter
GFP-cre
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Neuron-Specific AAV Promoter
• Synapsin 1 good for general neuronal expression
• Hioki H, Nakamura H, Okunomiya T, Ohira K, Nakamura K, Kuroda M et al (2007) Efficient
gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene
Therapy 14: 872-882.
https://www.ncbi.nlm.nih.gov/pubmed/?term=17361216
• Kugler S, Kilic E, Bahr M (2003) Human synapsin 1 gene promoter confers highly neuronspecific long-term transgene expression from an adenoviral vector in the adult rat brain
depending on the transduced area. Gene Therapy 10: 337-347.
https://www.ncbi.nlm.nih.gov/pubmed/?term=12595892
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Buying AAVs
• Need to fill out MTA forms (Material Transfer Agreement)
• https://uilo.ubc.ca/researchers/forms-and-templates
• Example of cost:
• From Laval with internal pricing 100 μl aliquot = $200
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BUT… before you buy/receive AAVs
• Need in place:
1. Biosafety Certificate (with Biohazard Assessment)
2. Animal Care Protocol for in vivo work
• These applications are completed on RISe (Research Information Services)
• https://www.rise.ubc.ca
• Might be amendment or new application
• Use other labs’ protocols as templates
• We (MacVicar lab) based ours off of Snutch protocol (thank you!)
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1. Biosafety Certificate
https://ors.ubc.ca/compliance-reporting/compliance-requirements/biosafety
• What’s included:
1) Study team
2) Funding sources
3) Procedures and Methodology
4) Recombinant Viral Vectors
5) Ethics
• Possibly add as amendment
• Also need Biohazard Risk Assessment (component of Biosafety Certificate)
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Biosafety for Viruses
• CL = containment Level
• RG = Risk Group
• AAVs:
• CL1 (RG1)
• Advantageous, less paperwork/equipment
• Lentivirus:
• Mainly CL2 (RG3)
• Or CL2 with CL3 Procedures
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Handling AAVs
• Keep AAVs on ice
• Aliquot in Class 2 Biosafety Hood
• Small volumes (eg. 5 μl in Eppendorfs, avoid repeated freezing-thawing)
• Can be freeze-thawed several times without loss in activity but should avoid
• Stanford Vector Core – Care and Handling of AAVs:
http://med.stanford.edu/gvvc/AAV.html
• Store at -80 oC
• Dispose of AAV + related material (eg. pipette tips) in 10 % bleach > 30 min
(in beaker)
• When handling PPE
• N95 respirator
• Gloves
• Lab coat
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Animal Care Protocol for in vivo work
• What’s included:
1) Study team
2) Study Dates and Funding
3) Animal Information & Type of Animal Review
4) Animal Information, Procedures, Justification
5) Animal Monitoring
6) Drugs and Chemicals
• Vet viewing/approval for members performing procedure
• Animal Care Committee meets once a month, can take a while to get new
application submitted:
• https://animalcare.ubc.ca/animal-care-committee/process/submission-deadlines
• But once application approved, can make amendments (faster)
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UBC Animal Care’s SOPs
• Cite UBC Standard Operating Procedures (SOPs):
• https://animalcare.ubc.ca/planning-your-research/sops-guidelines
• Relevant SOPs for stereotaxic surgery and injections (rats):
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Rat Stereotaxic Surgery (was not able to find online, e-mail Animal Care Committee for copy)
Rodent Survival Surgery (SOP ACC-02-2016)
Rodent Anesthesia (SOP ACC-01-2015)
Subcutaneous injection in mice and rats (TECH 11)
Local Anesthetic SOP (TECH 16)
Ketoprofen SOP (TECH 18)
SOP Euthanasia of Adult Rodents using CO2
• Monitoring sheets
• SOP 017 – Monitoring Policy
• https://animalcare.ubc.ca/animal-care-committee/policies-and-guidelines/017-animal-monitoringsheets-samples
• Grading system found online
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Rat Stereotaxic Surgery for Viral Injection
• UBC Rat Stereotaxic Surgery SOP
• New
• Supposed to be available online, but needed to e-mail
Animal Care Committee for copy
• Don’t know if there’s a Mouse Stereotaxic SOP
• Cetin A, Komai S, Eliava M, Seeburg PH, Osten P
(2007) Stereotaxic gene delivery in the rodent
brain. Nature Protocols 1: 3166-3173.
https://www.ncbi.nlm.nih.gov/pubmed/17406580
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