Gene Delivery to Muscle

Matthew L. Springer1, Thomas A. Rando1, Helen M. Blau1

1 Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.4
DOI:  10.1002/0471142905.hg1304s31
Online Posting Date:  February, 2002
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Abstract

The delivery of genes to skeletal muscle by myoblast implantation, DNA injection, or viral transduction has therapeutic applications for human neuromuscular and systemic disorders, many of which are now represented by transgenic or “knockout” mouse models. This unit describes the isolation and retroviral transduction of mouse myoblasts, the injection of myoblasts and plasmid DNA into mouse muscle, and histological methods for analyzing the recipient muscle. A procedure describing the injection of plasmid DNA into muscle with or without electric charge is also included.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Growth of Mouse Primary Myoblasts (Without Cell Sorting)
  • Alternate Protocol 1: Purification of Primary Myoblasts Using Cell Sorting
  • Basic Protocol 2: Infection of Primary Myoblasts with Retrovirus
  • Basic Protocol 3: Implantation of Myoblasts into Skeletal Muscle
  • Alternate Protocol 2: Direct Injection of Plasmid DNA into Muscle
  • Support Protocol 1: Isolation of lacZ‐Labeled Cells by Fluorescence‐Activated Cell Sorting (FACS)
  • Support Protocol 2: Histochemical Assessment of Implantation by X‐Gal and Hematoxylin/Eosin Staining of Cryosections
  • Reagents and Solution
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation and Growth of Mouse Primary Myoblasts (Without Cell Sorting)

  Materials
  • Neonatal mice, preferably 1 to 3 days old
  • 70% ethanol in squirt bottle
  • 1× phosphate buffered saline (PBS; see recipe), sterile
  • Collagenase/dispase/CaCl 2 solution (see recipe)
  • F‐10‐based primary myoblast growth medium (see recipe)
  • F‐10/DMEM‐based primary myoblast growth medium (see recipe)
  • Differentiation medium (optional; see recipe)
  • Sharp curved surgical scissors
  • Fine forceps (2 pairs)
  • Stereo dissecting microscope
  • Inverted microscope with phase optics
  • Sterile razor blades
  • Tissue culture plates
  • Tabletop centrifuge (e.g., IEC HN‐SII)
  • 35‐, 60‐, 100‐, and 150‐mm collagen‐coated tissue culture plates (see recipe)
  • Additional reagents and equipment for mammalian cell culture and freezing cells ( appendix 3G)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Purification of Primary Myoblasts Using Cell Sorting

  Materials
  • Primary myoblast culture (see protocol 1)
  • F‐10/DMEM‐based primary myoblast growth medium (see recipe)
  • Retroviral supernatants of choice (unit 12.5)
  • 800 µg/ml Polybrene (hexadimethrine bromide)
  • 6‐well or 60‐mm collagen‐coated tissue culture plates (see recipe)
  • Temperature‐controlled tabletop centrifuge equipped with microplate carrier (e.g., Beckman GPR with GH‐3.7 rotor)
  • Additional reagents and equipment for isolation and growth of primary myoblasts (see protocol 1)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Infection of Primary Myoblasts with Retrovirus

  Materials
  • Retrovirus‐infected cells to be injected (see protocol 3) sorted to isolate high expression (optional; see protocol 6)
  • Recipient mice
  • F‐10/DMEM‐based primary myoblast growth medium (see recipe)
  • 0.5% (w/v) BSA in 1× recipePBS (for PBS recipe), sterile
  • Metofane (methoxyflurane; optional)
  • 70% ethanol in squirt bottle
  • Tabletop centrifuge (e.g., IEC HN‐SII)
  • Gauze or cheesecloth
  • Inhalation chamber (e.g. glass chamber with grid or divider; optional)
  • 1‐ml syringes and 27‐G or 30‐G needles
  • Ear punch (optional)
  • Electric clippers or sharp curved surgical scissors
  • 50‐µl Hamilton syringe with metal needle adapter removed
  • Additional reagents and equipment for culturing retrovirally infected myoblasts (see protocol 3) and trypsinizing and counting cells ( appendix 3G)
NOTE: All instruments must be sterile and proper sterile technique must be used accordingly.

Basic Protocol 3: Implantation of Myoblasts into Skeletal Muscle

  Materials
  • 200 mM FDG (100×; Molecular Probes) in 1:1 DMSO/H 2O (store at −20°C in the dark)
  • lacZ‐expressing cells to be sorted (see protocol 3) and control cells not expressing lacZ
  • recipeFACS staining buffer (see recipe), room temperature to 37°C
  • recipeFACS stop buffer (see recipe), ice‐cold
  • F‐10‐ or F‐10/DMEM‐based primary myoblast growth medium
  • Tabletop centrifuge (e.g., IEC HN‐SII)
  • Fluorescence‐activated cell sorter (e.g., Becton Dickinson FACStar)
  • Collagen‐coated tissue culture plates (see recipe)
  • Additional reagents and equipment for trypsinization of cells ( appendix 3G)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Direct Injection of Plasmid DNA into Muscle

  Materials
  • Mouse with transplanted myoblasts (see protocol 4)
  • OCT embedding compound (Miles Labs)
  • Isopentane (2‐methylbutane, store at 4°C)
  • Liquid nitrogen
  • X‐gal fixative solution (see recipe)
  • recipe1× phosphate‐buffered saline (PBS; see recipe)
  • 40 mg/ml X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside) in dimethyl formamide (store up to 6 months at −20°C)
  • X‐gal staining solution (see recipe)
  • Aqueous mounting medium (Airvol or Gelvatol, Air Products and Chemicals)
  • Hematoxylin (Harleco brand from EM Science; filter before use or skim gold film off surface with Kimwipe)
  • Scott's water (see recipe)
  • Eosin Y (Sigma)
  • 95% ethanol
  • 100% ethanol
  • 1:1 mixture of xylene and 100% ethanol
  • Xylene
  • Pro‐Texx mounting medium (Baxter)
  • Dissecting board with bent pins
  • Fine forceps
  • Sharp curved surgical scissors and heavier 2‐in. scissors
  • Plastic cryomold (biopsy‐size, 10 × 10 × 5 mm; Miles Labs)
  • Indelible ink marking pen
  • Metal beaker
  • Dewar flask
  • String
  • Ring‐stand post with clamps
  • Heavy metal tongs
  • Flat piece of dry ice inside a styrofoam box with lid
  • Cryostat
  • Gelatin‐coated or electrostatically charged microscope slides (see recipe)
  • Hydrophobic slide marker (PAP pen, Research Products International)
  • Glass slide rack
  • Moist chamber (unit 4.3)
  • Coverslips (24 mm × 50 mm)
  • Tungsten color slide film (e.g., Kodak Ektachrome 160 T)
  • High‐quality compound microscope fitted with D.I.C. (Nomarski) optics, bright‐field optics, and photography system
  • Glass slide staining chambers with lids (at least 12)
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Figures

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Literature Cited

Literature Cited
   Aihara, H. and Miyazaki, J. 1998. Gene transfer into muscle by electroporation in vivo. Nature Biotechnology 16:867‐870.
   Barr, E. and Leiden, J.M. 1991. Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254:1507‐1509.
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