Retinal Gene Delivery by rAAV and DNA Electroporation

Aditya Venkatesh1, Shan Ma1, Fernanda Langellotto2, Guangping Gao3, Claudio Punzo3

1 Department of Ophthalmology, University of Massachusetts Medical School, Worcester, Massachusetts, 2 Current address: Children's Hospital, Harvard Medical School, Boston Massachusetts, 3 Co‐corresponding authors
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14D.4
DOI:  10.1002/9780471729259.mc14d04s28
Online Posting Date:  February, 2013
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Abstract

Ocular gene therapy is a fast‐growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye‐specific genes tend to be more common. To date, over 40 eye‐specific genes have been identified harboring mutations that lead to blindness. Gene therapy with recombinant adeno‐associated virus (rAAV) holds the promise to treat patients with such mutations. However, proof‐of‐concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV‐mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, and subsequent tissue processing methods are described. Curr. Protoc. Microbiol. 28:14D.4.1‐14D.4.32. © 2013 by John Wiley & Sons, Inc.

Keywords: retina; eye; gene therapy; gene transfer

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Delivery of rAAV by Subretinal and Intravitreal Injection into Eyes of Newborn Mice
  • Basic Protocol 2: Delivery of Plasmid DNA by Subretinal Injection into Eyes of Newborn Mice
  • Basic Protocol 3: Delivery of rAAV by Subretinal and Intravitreal Injection into the Eye of Adult Mice
  • Alternate Protocol 1: Subretinal and Intravitreal Injection with Hamilton Syringes
  • Basic Protocol 4: Fundoscopy Examination to Monitor Infection Area
  • Basic Protocol 5: Whole‐Mount Immunofluorescence Analyses
  • Basic Protocol 6: Whole‐Mount In Situ Hybridizations
  • Support Protocol 1: Dissection and Tissue Processing for Cryosectioning
  • Support Protocol 2: Dissection and Tissue Processing for Paraffin Sectioning
  • Basic Protocol 7: Immunofluorescence Analysis on Cryo‐ or Paraffin Sections
  • Basic Protocol 8: In Situ Hybridization Analysis on Cryo‐ or Paraffin Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Delivery of rAAV by Subretinal and Intravitreal Injection into Eyes of Newborn Mice

  Materials
  • Naturally delivered newborn mouse pups (0‐ to 1‐day old) of any strain
  • Buprenorphine
  • Virus: rAAV can be produced in‐house (unit 14.1), or obtained through a local vector core or via a commercial manufacturer
  • 0.05% (w/v) Fast Green solution (Sigma, cat. no. F‐7252, or equivalent)
  • Betadine
  • 70% ethanol
  • Insulin injection needles
  • Weight trays appropriate for mice
  • Heating plate/mat
  • Rubber gloves
  • Ice bucket
  • Glass needles (Humagen Custom O from Origio, http://www.origio.com/)
  • Microloader (pipet tips to load the glass needle; Eppendorf, cat. no. 93001007, or equivalent)
  • Dissecting microscope with appropriate light source
  • Cotton swabs
  • Injection pump (FemtoJet from Eppendorf)
  • 30‐G disposable needles
  • Forceps (student Dumont no. 5 work well)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 2: Delivery of Plasmid DNA by Subretinal Injection into Eyes of Newborn Mice

  Materials
  • Plasmid DNA (concentration, 1 to 5 µg/µl)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Square‐filed electroporator (e.g., Harvard Apparatus, cat. no. 450052)
  • Tweezer electrodes (e.g., Harvard Apparatus, cat. no. 405166)
  • Additional reagents and equipment for delivery of rAAV by subretinal and intravitreal injection into eyes of newborn mice ( protocol 1)

Basic Protocol 3: Delivery of rAAV by Subretinal and Intravitreal Injection into the Eye of Adult Mice

  Materials
  • Adult mouse of any age and strain
  • Ketamine
  • Xylazine
  • Corneal lubricant ointment
  • Betadine
  • 70% ethanol
  • Buprenorphine
  • Dissecting microscope with appropriate light source
  • Glass needles (Humagen Custom O from Origio, http://www.origio.com/)
  • Heating plate/mat
  • Additional reagents and equipment for delivery of rAAV by subretinal and intravitreal injection into eyes of newborn mice ( protocol 1)

Alternate Protocol 1: Subretinal and Intravitreal Injection with Hamilton Syringes

  • Virus or DNA
  • Phosphate‐buffered saline (PBS; appendix 2A; if using DNA)
  • Hamilton syringe with blunt needle
  • Square‐filed electroporator (e.g., Harvard Apparatus, cat. no. 450052; if using DNA)
  • Tweezer electrodes (e.g., Harvard Apparatus, cat. no. 405166; if using DNA)
  • Hamilton syringe with blunt needle
  • Additional reagents and equipment for delivery of rAAV by subretinal and intravitreal injection into eyes of newborn mice ( protocol 1)

Basic Protocol 4: Fundoscopy Examination to Monitor Infection Area

  Materials
  • Injected (Basic Protocol protocol 11 to protocol 33) mouse of any strain, at least 2 weeks post‐injection
  • Phenylephrine (Akorn, cat. no. NDC: 17478‐200‐12, http://www.akorn.com)
  • Tropicamide (Akorn, cat. no. NDC: 17478‐101‐12, http://www.akorn.com)
  • Ketamine
  • Xylazine
  • Funduscope equipment (Micron III; Phoenix Research Laboratories Inc., http://www.phoenixreslabs.com/):
    • Funduscope camera
    • Appropriate light source
    • Platform to position mouse
    • Computer system and software
  • Heating plate/mat
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 5: Whole‐Mount Immunofluorescence Analyses

  Materials
  • Injected (Basic Protocol protocol 11 to protocol 33) or noninjected mouse of any strain
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% PFA/PBS: 4% paraformaldehyde in PBS, pH 7.4
  • PBT: PBS with 0.3% (v/v) Triton X‐100
  • PBTB: PBT (see above) with 5% (v/v) bovine serum albumin (BSA)
  • Primary and secondary antibodies
  • Appropriate reagents for color development of secondary antibody
  • Mounting medium: e.g., Gel Mount, Fluoromount (Sigma‐Aldrich)
  • Clear nail polish (optional)
  • CO 2 chamber for euthanasia (see Donovan and Brown, )
  • Forceps (student Dumont no. 5 work well)
  • 6‐cm Petri dish
  • Dissecting scope with light source
  • Small spring scissor
  • Cover glasses
  • Glass slides
  • Fluorescence upright or inverted microscope
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Basic Protocol 6: Whole‐Mount In Situ Hybridizations

  Materials
  • 10× transcription buffer (supplied with the RNA polymerase)
  • 10× nucleotide labeling mix (Roche; DIG labeling mix, cat. no. 11277073910)
  • Template DNA: linearized plasmid DNA or PCR product; not shorter than 200 bp, ideally between 500 and 1000 bp
  • RNase inhibitor (Roche, cat. no. 03335399001)
  • RNA polymerase (Roche): T7, T3, or SP6, depending on the PCR primers used to generate the PCR product; make sure to take the correct RNA polymerase to synthesize antisense RNA
  • H 2O, DEPC‐treated (see recipe for DEPC treatment)
  • RNase‐free DNase (Roche, cat. no. 04716728001)
  • TE buffer, pH 8 ( appendix 2A), DEPC‐treated (see recipe for DEPC treatment)
  • 4 M LiCl, prepared with DEPC‐treated H 2O (see recipe for DEPC treatment)
  • 100% and 70% ethanol
  • Hybridization buffer (HB; for whole‐mount in situ hybridization; see recipe)
  • 4% PFA/PBS: 4% paraformaldehyde in PBS, pH 7.4
  • PBS/Tween: 0.1% (v/v) Tween‐20 in phosphate buffered saline (PBS; appendix 2A) that has been treated with DEPC (see recipe for DEPC treatment)
  • 100% methanol
  • 10 mg/ml proteinase K stock (store at −20°C)
  • 20× SSC ( appendix 2A)
  • 10 mM PIPES/500 mM NaCl, pH 6.8
  • MAB: 100 mM maleic acid, pH 7.5/150 mM NaCl
  • Blocking reagent (Roche, cat. no. 11096176001)
  • MABT: MAB (see above) containing 0.1% (v/v) Tween‐20
  • Antibody: α‐digoxigenin antibody coupled to alkaline phosphatase (α‐DIG‐AP)
  • 100 mM Tris⋅Cl, pH 9.5 ( appendix 2A) containing 0.1% (v/v) Tween‐20
  • Staining solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 10 mM EDTA
  • Glycerol
  • Glass scintillation vials
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), for dissecting the retina (see protocol 6, steps 1 to 5), and for flat mounting of the retina ( protocol 1, step 17)

Support Protocol 1: Dissection and Tissue Processing for Cryosectioning

  Materials
  • 4% PFA/PBS: 4% paraformaldehyde in PBS, pH 7.4
  • Phosphate‐buffered saline, pH 7.4 ( appendix 2A)
  • 10%, 20%, and 30% sucrose in PBS
  • OCT embedding medium (Tissue‐Tek)
  • Dry ice/isopropanol bath
  • Dissecting microscope with light source
  • Rocker or Nutator
  • 6‐cm Petri dish
  • Forceps (student Dumont no. 5 work well)
  • Small spring scissor
  • Embedding molds
  • Cryostat
  • Additional reagents and equipment for removal of the mouse eyeball for whole‐mount analysis ( protocol 6, steps 1 and 2) and antibody staining (optional; protocol 10)

Support Protocol 2: Dissection and Tissue Processing for Paraffin Sectioning

  Materials
  • Tissue (harvested from step 2 or step 5 of protocol 8)
  • 4% PFA/PBS: 4% paraformaldehyde in PBS, pH 7.4
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 25%, 50%, 75%, AND 100% ethanol in PBS
  • Xylene
  • Paraffin
  • Incubator/oven (controllable up to 65°C for paraffin)
  • Embedding molds
  • Microtome for paraffin sections
  • 45°C water bath
  • Glass slides pretreated for paraffin sections
  • Heating plate

Basic Protocol 7: Immunofluorescence Analysis on Cryo‐ or Paraffin Sections

  Materials
  • Tissue: cryosections ( protocol 8) or paraffin sections ( protocol 9)
  • PBT: Phosphate‐buffered saline PBS with 0.3% (v/v) Triton X‐100
  • PBTB: PBT (see above) with 5% (v/v) bovine serum albumin (BSA)
  • Primary and secondary antibodies
  • Mounting medium: e.g., Gel Mount, Fluoromount (Sigma‐Aldrich)
  • Nuclear DAPI stain (Sigma‐Aldrich, cat. no. D‐9542)
  • Mounting media: Gel mount, Fluoromount, etc.
  • Humidified incubation chamber: slide box with wet paper towels on the bottom; seal box with plastic tape
  • Cover glasses

Basic Protocol 8: In Situ Hybridization Analysis on Cryo‐ or Paraffin Sections

  Materials
  • Tissue: cryosections ( protocol 8) or paraffin sections ( protocol 9)
  • 4% PFA/PBS: 4% paraformaldehyde in PBS, pH 7.4
  • PBS/Tween: 0.1% (v/v) Tween‐20 in phosphate buffered saline (PBS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mg/ml proteinase K stock (store at −20°C)
  • Acetic acid anhydride
  • 1 M triethanolamine (TEA) stock, pH 8.0)
  • Hybridization buffer (HB; for section in situ hybridization; see recipe)
  • RNA probe ( protocol 7)
  • 20× SSC ( appendix 2A)
  • Formamide (molecular biology grade, 99% pure)
  • TNE (see recipe)
  • RNase A (Sigma‐Aldrich, cat. no. R‐4875 or equivalent)
  • MAB: 100 mM maleic acid, pH 7.5/150 mM NaCl
  • MABT: MAB (see above) containing 0.1% (v/v) Tween‐20
  • HISS (heat‐inactivated sheep serum)
  • Antibody: α‐digoxigenin antibody coupled to alkaline phosphatase (α‐DIG‐AP)
  • NTMT, pH 9.5 (see recipe)
  • Staining solution (see recipe)
  • Mounting medium (e.g., Gelvatol; see recipe)
  • Cover glasses or home‐made coverslips from polyethylene bags: cut plastic bags with ruler and razor blade into small pieces the size of a cover glass and store them in a petri dish
  • Humidified incubation chamber: slide box with wet paper towels on the bottom; seal box with plastic tape
  • 65°C hybridization oven
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Figures

Videos

Literature Cited

Literature Cited
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