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Databinge AAV PresentationMatthieu 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
2
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
3
https://www.addgene.org/mol-bio-reference/plasmid-background/
Addgene
• Non-profit plasmid repository • 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
• Eg. mitochondrial calcium
2. Sort + Filters to narrow down• Eg. popularity + mammalian expression
3. Select plasmid• Eg. CMV-mito-R-GECO1 (#46021)
4. Literature Search• 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: 1888-1891.
https://www.ncbi.nlm.nih.gov/pubmed/?term=219037794
https://www.addgene.org/recipient-instructions/streak-plate/
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
5
Isolate pDNA
Plasmid from Addgene
Transfect Transduce(eg. in mammalian cultures, in vivo)
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/cell-culture/transfection/transfection-reagent-application-table.html
• Lipid Nanoparticles (LNPs):http://www.liposomes.ca• Dr. Pieter Cullis @ UBC
• Packaging size limit?
6
Example of transfection protocol(Thermo Fisher Scientific)
Transduction with Viruses
• Advantages:• Efficient
• Disadvantages• More paperwork
• Packaging size limits
7
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
9
Adeno-Associated Viruses (AAVs)
• Wild-type AAV: • 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
10
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
• Many serotypes
11
http://ppn.med.sc.edu/Adeno-associated.Virus.asp
• Produce recombinant AAVs by co-transfecting 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
Recombinant AAVs (rAAVs)
12
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
13
Addgene
AAV Serotypes
14
UPenn
AAV Serotypes
?Wide expression
Retrograde transport
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) Comparitiveanalyses 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
16
Aschauer DF et al. (2013) Paper
17Expression Inflammatory Response
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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AAV1.Syn.flex.ChR2-GFP.WPRE.SV40
“.” dot to separate sequences“-” dash for fusion proteinsserotype
Sequences of interest(will be transcripted/translated to the precious protein wanted)
Associated sequences(won’t be expressed but will impact the expression of the sequences of interest)
Promoter
Extrinsic Specificity(Optional)
AAV1.Syn.cre
AAV Grammar
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Promoters (Mammalian Applications)• General expression
• CMV: Cytomegalovirus• CBA (emCBA: enhance mini): chicken β actin (CI: CBA intron?)
• CMV enhancer + CBA = CAG (or CB7) synthetic promoter, CAGGS(?)
• EF1a: Human elongation factor-1 alpha. • PGK1: phosphoglycerate kinase• Ubc: ubiquitin C
• Specific expression• 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)
20https://www.addgene.org/tetracycline/
CaMK2a-tTA mouse(jax 003010)
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.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651700/
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
22
Common gene of interest and fusion proteins/sequence
• Functional Neurophotonics and other genetic engineering• 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, …
23
Cre/Lox RecombinationCre 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 (Double-floxed Inverse Orientatio) or or double flox (dflox)• flex-frwd: cre inactivation by inversion• e.g. AAV1.Syn.Flex.GCaMP6f.WPRE.SV40
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CAG stop tdTomato
CAG tdTomatostop
LSL:
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 (INTronicRecombinase Sites Enabling Combinatorial Targeting, Fenno et al. 2014 Nature Methods): Bolean logic• 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/viral-service/aav-prep/
26
Serotypes mostly 1, 5, 9Synapsin promoter
CaMPARI: Switchable GCaMP
WPRE: enhancer
polyA sequence = stop
Different GCaMP
CAG promoter
CaMK2 promoter
NES: nuclear localization
jrGECO1a: red GCaMP6
jRCaMP1a: red GCaMP6
27
iGluSnFR: glutamate sensor
GFAP promoter
Twitch2B: FRET calcium indicator
mRuby2: red reporter
2A: self-cleaving GfaABC1D GFAP promoter
Lck=membrane localization
Jaws-KGC: red activated chloride pump targeted to membrane
28
hGH,bGH polyA
tdTomato, GFP fusion protein
29EF1a promoter
30
Flex inversion
31
gene knock-out: “floxing”
2A cleavage
DIO=flex
CBA promoter
32
33
Cre express under CMV promoter
GFP-cre
34
35
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 neuron-specific 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
36
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
37
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!)
38
1. Biosafety Certificate
https://ors.ubc.ca/compliance-reporting/compliance-requirements/biosafety
• What’s included:1) Study team 2) Funding sources3) Procedures and Methodology 4) Recombinant Viral Vectors 5) Ethics
• Possibly add as amendment
• Also need Biohazard Risk Assessment (component of Biosafety Certificate)
39
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
40
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
41
Animal Care Protocol for in vivo work
• What’s included:1) Study team 2) Study Dates and Funding 3) Animal Information & Type of Animal Review4) Animal Information, Procedures, Justification5) Animal Monitoring6) 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)
42
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):
• 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-monitoring-sheets-samples
• Grading system found online
43
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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