Ex Vivo and In Vivo Gene Delivery to the Brain

Jaana Suhonen1, Jasodhara Ray1, Ulrike Blömer1, Fred H. Gage1, Brian Kaspar2

1 The Salk Institute, La Jolla, California, 2 Columbus Children's Research Institute, Columbus, Ohio
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.3
DOI:  10.1002/0471142905.hg1303s51
Online Posting Date:  November, 2006
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Abstract

This unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.

Keywords: Nervous system; transplantation; genetically modified cells; recombinant virus; gene therapy

     
 
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Table of Contents

  • Basic Protocol 1: Implantation of Genetically Modified Cells into the Adult Rat Brain
  • Basic Protocol 2: Implantation of Genetically Modified Cells into the Neonatal Rat Brain
  • Basic Protocol 3: Implantation of Genetically Modified Cells into the Fetal Rat Brain
  • Basic Protocol 4: Implantation of Collagen‐Embedded Genetically Modified Fibroblasts into a Cavity in the Adult Rat Brain
  • Basic Protocol 5: Direct Injection of Recombinant Viral Vectors into the Rat Brain
  • Support Protocol 1: Preparation of Genetically Modified Cells for Grafting
  • Support Protocol 2: Transcardial Perfusion of Adult and Neonatal Rats
  • Support Protocol 3: Sectioning of the Rat Brains in Freezing Sledge Microtome
  • Support Protocol 4: Thionine Staining of Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Implantation of Genetically Modified Cells into the Adult Rat Brain

  Materials
  • Culture of genetically engineered cells (see protocol 6 and Critical Parameters)
  • Adult rat (see Critical Parameters)
  • recipeAnesthetic solution (see recipe)
  • Betadine solution (10% povidone iodine, J.A. Webster)
  • Opthalmic ointment (Mycitracin, Upjohn)
  • 30% (v/v) hydrogen peroxide
  • Antibiotic powder (Neo‐Predef, Upjohn)
  • Rat brain atlas (Paxinos and Watson, )
  • Ear punch (Fisher) or ear tags (Harvard Bioscience)
  • Stereotaxic frame with electrode manipulator and clamp for Hamilton syringe (Small Animal Stereotaxic, model no. 900, and 45° tip non‐rupture ear bars, model no. 955, both from David Kopf Instruments)
  • Surgical instruments:
    • Scalpel with rounded no. 10 blades
    • Bulldog clamps
    • Swabs
    • Medium forceps
    • 7‐mm wound clips
    • Wound‐clip applicator
  • Dental drill with size 2 carbide burr
  • 26‐G needles
  • 5‐µl Hamilton syringe and needles (≤26‐G; see Critical Parameters)
  • Additional reagents and equipment for preparing suspension of genetically engineered cells ( protocol 6)
NOTE: All surgical instruments must be sterile and all procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 2: Implantation of Genetically Modified Cells into the Neonatal Rat Brain

  Materials
  • Culture of genetically engineered cells (see protocol 6 and Critical Parameters)
  • Newborn rats (P0‐P2 pups)
  • 100% ethanol
  • Antibiotic powder (Neo‐Predef, Upjohn)
  • Surgical glue (Vetbond Tissue Adhesive no. 1469, 3M or J.A. Webster)
  • Operating microscope
  • Surgical instruments:
    • Scalpel with rounded no. 10 and sharp no. 11 blades and handles
    • Swabs
    • Microdissecting forceps
  • Neonatal rat brain atlas (Paxinos et al., ; Altman and Bayer, )
  • 5‐µl Hamilton syringe and 26‐G needles or glass micropipet
  • Stereotaxic frame with electrode manipulator and clamp for Hamilton syringe (Small Animal Stereotaxic, model no. 900, and 45° tip non‐rupture ear bars, model no. 955; both from David Kopf Instruments)
  • 37°C heating pad or heat lamp
  • Additional reagents and equipment for preparing suspension of genetically engineered cells ( protocol 6)
NOTE: All surgical instruments must be sterile and all procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 3: Implantation of Genetically Modified Cells into the Fetal Rat Brain

  Materials
  • Culture of genetically engineered cells (see protocol 6 and Critical Parameters)
  • Timed pregnant rat (E14 and up)
  • recipeAnesthetic solution (see recipe)
  • 75% ethanol
  • Ophthalmic ointment (Mycitracin, Upjohn)
  • Antibiotic powder (Neo‐Predef, Upjohn)
  • 37°C hot plate
  • Sterile towels
  • Surgical instruments:
    • Sharp scissors
    • Tissue forceps
    • Needle holder
    • Suture thread (5–0 chromic catgut, 5–0 braided silk)
    • Swabs
  • Optic fiber (Donlan Jenner Industries)
  • Prenatal rat brain atlas (Altman and Bayer, )
  • 5‐µl Hamilton syringe and 30‐G needle or glass micropipet
  • Additional reagents and equipment for preparing suspension of genetically engineered cells ( protocol 6)
NOTE: All surgical instruments must be sterile and all procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 4: Implantation of Collagen‐Embedded Genetically Modified Fibroblasts into a Cavity in the Adult Rat Brain

  Materials
  • Culture of genetically engineered fibroblasts (see protocol 6 and Critical Parameters)
  • recipeComplete DMEM/10% FBS medium (see recipe)
  • 0.1 M NaOH
  • recipe0.3% collagen (see recipe)
  • Adult rat (see Critical Parameters)
  • 1.5‐ml conical screw cap tubes (Nalgene)
  • 37°C, 10% CO 2 incubator
  • Stereotaxic frame (Small Animal Stereotaxic, model no. 900, and 45° tip ear bars, model No. 955, both from David Kopf Instruments)
  • Surgical instruments:
    • Scalpel with rounded no. 10 blades
    • Bulldog clamps
    • Swabs
    • Medium and fine forceps
    • 7‐mm wound clips
    • Wound‐clip applicator
  • Rat brain atlas (Paxinos and Watson, )
  • Dental drill with size 2 carbide burr
  • 27‐G needles
  • Modified Pasteur pipet attached to vacuum source (see step annotation)
  • Hydrated gel foam (Upjohn)
  • Additional reagents and equipment for preparing suspension of genetically engineered cells ( protocol 6) and anesthetizing and preparing rat for surgery ( protocol 1)
NOTE: All solutions and equipment coming into contact with cells must be sterile and proper sterile technique should be used accordingly (see appendix 3G). All surgical instruments must be sterile and all procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 5: Direct Injection of Recombinant Viral Vectors into the Rat Brain

  Materials
  • Adult rat
  • recipeAnesthetic solution (see recipe)
  • Betadine solution (10% povidone iodine, J.A. Webster)
  • Ophthalmic ointment (Mycitracin, Upjohn)
  • Recombinant virus suspension (see and Chapter 12) in PBS, TBS or HEPES‐buffered saline (HBS) ( appendix 2D)
  • Antibiotic powder (Neo‐Predef, Upjohn)
  • Rat brain atlas (Paxinos and Watson, )
  • Ear punch (Fisher) or ear tags (Harvard Bioscience)
  • Stereotaxic frame (Small Animal Stereotaxic, model no. 900, and 45° tip non‐rupture ear bars, model no. 955, both from David Kopf Instruments)
  • Surgical instruments:
    • Scalpel with rounded no. 10 blades
    • Bulldog clamps
    • Swabs
    • Medium forceps
    • 7‐mm wound clips (or sutures)
    • Wound‐clip applicator
  • Dental drill with size 2 carbide burr
  • 26‐G needles
  • 5‐µl Hamilton syringe with beveled 26‐G needle

Support Protocol 1: Preparation of Genetically Modified Cells for Grafting

  Materials
  • Recombinant fibroblasts or neural progenitor cells transfected with retroviral (unit 12.5) or lentiviral (unit 12.10) vector (also see Commentary)
  • recipeDMEM/10% FBS or recipeDMEM/F12/N2/FGF medium (see reciperecipes), 37°C
  • Phosphate‐buffered saline (PBS; appendix 2D), 37°C
  • ATV trypsin (Irvine Scientific)
  • recipeDulbecco's phosphate‐buffered saline (D‐PBS, see recipe), 37°C
  • recipeD‐PBS (see recipe) containing 20 ng/ml FGF‐2 (Pepro Tech or R&D Systems)
  • 75‐cm2 tissue culture flasks or 10‐cm tissue culture plates
  • IEC Clinical centrifuge (Fisher)
  • Coulter counter (Coulter)
  • Additional reagents and equipment for tissue culture ( appendix 3G)
NOTE: All materials used for culturing cells should be sterile and the cells should be handled in a laminar flow hood until their final suspension in appropriate medium for grafting. Prewarm all solutions and media to be used for culturing to 37°C.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Transcardial Perfusion of Adult and Neonatal Rats

  Materials
  • Rat ( protocol 1, protocol 22, protocol 33, protocol 44, or protocol 55)
  • recipeAnesthetic solution (see recipe)
  • 0.9% NaCl (ice‐cold)
  • recipe4% paraformaldehyde (see recipe) containing 0.1% (w/v) glutaraldehyde (optional)
  • 30% (w/v) sucrose/0.1 M sodium phosphate, pH 7.3 (see recipes for sodium phosphate in appendix 2D), 4°C
  • Down‐draft table (Jewett Corporation)
  • Surgical instruments:
    • Sharp‐blunt scissors
    • Tissue forceps
    • Crile forceps
    • Bone rongeur
  • Masterflex peristaltic pump (Cole‐Parmer) fitted with:
    • C‐FLEX tubing (Cole‐Parmer)
    • Cannula (18‐G needle with beveled tip cut off to give rounded end)
NOTE: All perfusions are done on a down‐draft table. Because paraformaldehyde is toxic, remove the brain from the perfused animal in a chemical hood. Use safety glasses and mask. See appendix 2A for guidelines in working with hazardous chemicals.

Support Protocol 3: Sectioning of the Rat Brains in Freezing Sledge Microtome

  Materials
  • Rat brain (See protocol 8)
  • OCT compound (Tissue‐Tek II; Miles Labs)
  • Powdered dry ice
  • recipeTCS (see recipe)
  • Freezing sledge microtome (Leica)
  • No. 0 paint brush
  • 24‐ to 96‐well tissue culture plates (Corning)
  • 100‐mm petri dishes
  • recipeGelatin‐coated slides (see recipe)

Support Protocol 4: Thionine Staining of Tissue Sections

  Materials
  • Slides containing fixed, sectioned brain tissue (see protocol 8)
  • Chloroform
  • 100%, 95%, 80%, 70%, and 50% (v/v) ethanol
  • 0.25% thionine stain (see recipe)
  • 2‐propanol
  • Xylene
  • Pro‐Texx mounting medium (Baxter)
  • Coplin jars
  • Coverslips
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Figures

Videos

Literature Cited

   Akli, S., Caillaud, C., Vigne, E., Stratford‐Perricaudet, L.D., Ponaru, L., Perricaudet, M., Kahn, A., and Peschanski, M.R. 1993. Transfer of a foreign gene into the brain using adenovirus vectors. Nature Genet. 3:224‐228.
   Altman, J. and Bayer, S. 1995. Atlas of Prenatal Rat Brain Development. CRC Press, Boca Raton, Fla.
   Bajocchi, G., Feldman, S.H., Crystal, R.G., and Mastrangeli, A. 1993. Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors. Nature Genet. 3:229‐234.
   Burger, C., Gorbatyuk, O.S., Velardo, M.J., Peden, C.S., Williams, P., Zolotukhin, S., Reier, P.J., Mandel, R.J., and Muszyczka, N. 2004. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10:302‐317.
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   Doering, L.C. and Chang, P.L. 1991. Expression of novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system. J. Neurosci. Res. 29:292‐298.
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   Kaspar, B.K., Vissel, B., Bengoechea, T., Crone, S., Randolph‐Moore, L., Muller, R., Brandon, E.P., Schaffer, D., Verma, I.M., Lee, K.F., Heinemann, S.F., and Gage, F.H. 2002. Adeno‐associated virus effectively mediates conditional gene modification in the brain. Proc. Natl. Acad. Sci. U.S.A. 99:2320‐2325.
   Kawaja, M.D., Fisher, L.J., Schinstine, M., Jinnah, H.A., Ray, J., Chen, L.S., and Gage, F.H. 1992. Grafting genetically modified cells within the rat central nervous system: Methodological considerations. In Neural Transplantation: A Practical Approach, (S.B. Dunnett and A. Bjorklund eds.) pp. 20‐55. Oxford University Press, New York.
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Key References
   Kawaja et al. 1992. See above.
  This review article examines different methods for gene delivery to various cell types that may be used for gene therapy in the central nervous system and the methods used to graft these cells in the rat brain. The methods for assessing transgene expression from genetically modified cells before and after grafting are also discussed.
   Kriegler, M. 1990. Gene Transfer and Expression: A Laboratory Manual. W.H. Freeman, New York.
  This manual describes the background information and methods for gene transfer, as well as methods for monitoring the transfer and expression of the transgenes.
   Ray and Gage, 1993. See above
  This review article describes the production of packaging cell lines with retroviral vectors and the production of genetically modified cells using recombinant virus.
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