Gene Delivery to the Airway

Nicholas W. Keiser1, John F. Engelhardt1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.9
DOI:  10.1002/0471142905.hg1309s78
Online Posting Date:  July, 2013
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Abstract

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts, as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. Curr. Protoc. Hum. Genet. 78:13.9.1‐13.9.41. © 2013 by John Wiley & Sons, Inc.

Keywords: genetics and genomics; gene therapy

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Human Primary Airway Epithelial Cells
  • Basic Protocol 2: Transduction of Primary Airway Epithelial Cells
  • Basic Protocol 3: Generation of Polarized Primary Airway Epithelial Monolayers
  • Alternate Protocol 1: Generation of Polarized Airway Epithelial Monolayers from Immortalized Cell Lines
  • Basic Protocol 4: Gene Transfer to Polarized Airway Epithelia
  • Basic Protocol 5: Generation of Human Tracheal Xenografts
  • Alternate Protocol 2: Generation of Newborn Ferret Tracheal Xenografts
  • Basic Protocol 6: Gene Transfer to Human Tracheal Xenografts
  • Basic Protocol 7: In Vivo Gene Delivery to the Lung
  • Support Protocol 1: Harvesting of Human Tracheal Xenografts for Morphologic Analysis to Evaluate Transgene Expression
  • Support Protocol 2: Histochemical Detection of β‐Galactosidase Transgene Activity
  • Support Protocol 3: Immunohistochemical Detection of β‐Galactosidase Transgene Activity
  • Support Protocol 4: Fluorescent Detection of Green Fluorescent Protein to Evaluate Transgene Expression
  • Support Protocol 5: Histochemical Detection of Alkaline Phosphatase Transgene Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Human Primary Airway Epithelial Cells

  Materials
  • Human lung (keep on ice)
  • Ice
  • Media A, B, and C (see reciperecipes)
  • Medium A (see recipe) supplemented with 0.1% (w/v) protease type XIV (e.g., Sigma), 4°C
  • Fetal bovine serum (FBS; appendix 3G)
  • Ham's F‐12 medium (Life Technologies) with and without 10% FBS
  • 0.1% trypsin/EDTA (Life Technologies)
  • Trypsin inhibitor buffer (see recipe)
  • Cryopreservation medium: medium C (see recipe) containing 10% DMSO and 10% FBS, 4°C
  • Tissue culture hood
  • Dissecting equipment including:
    • Forceps
    • Scalpel
    • Hemostat
  • 100‐ and 150‐mm tissue culture dishes (uncoated plastic)
  • 15‐and 50‐ml conical centrifuge tubes
  • Platform rocker
  • Tabletop centrifuge
  • 3‐cm2 piece of 500‐µm nichrome or copper wire mesh, sterile
  • 2‐ml cryovials (e.g., Nunc)
  • Additional reagents and equipment for culturing of mammalian cells and counting viable cells by trypan blue exclusion ( appendix 3G)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Transduction of Primary Airway Epithelial Cells

  Materials
  • 8 mg/ml polybrene (Sigma) in H 2O (store in aliquots at −20°C)
  • Primary airway epithelial cells (see protocol 1; freshly isolated cells are needed for retroviral infection; once‐passaged cells may be used for adeno‐associated virus infection)
  • Ham's F‐12 medium (Life Technologies)
  • Medium C (see recipe)
  • 0.45‐µm syringe filter
  • 100‐mm tissue culture dishes
  • 12,000‐ to 14,000‐Da MWCO dialysis membrane (Life Technologies)
  • Additional reagents and equipment for culturing of mammalian cells and producing retroviral or lentivirus vectors (units 12.5& 12.10) or producing and purifying adeno‐associated viral vectors (unit 12.9)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Generation of Polarized Primary Airway Epithelial Monolayers

  Materials
  • Glacial acetic acid
  • Collagen, Type IV, acid‐soluble, from human placenta (Sigma Type VI)
  • Phosphate‐buffered saline (PBS: appendix 2D)
  • 5% serum airway medium (see recipe)
  • Ussing chamber culture medium (see recipe)
  • 0.2‐µm filter (Millipore Millex GS or equivalent Nalgene filter)
  • 12‐mm Millicell‐HA culture plate inserts (Millipore)
  • 24‐well tissue culture plates
  • Additional reagents and equipment for culturing of mammalian cells and counting viable cells by trypan blue exclusion ( appendix 3G) and generating airway epithelial cells (see protocol 1)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 1: Generation of Polarized Airway Epithelial Monolayers from Immortalized Cell Lines

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • NuLi or CuFi cells (University of Iowa Cells and Tissue Core, Joseph Zabner, Director)
  • BEGM medium (see recipe)
  • 0.25% Trypsin‐EDTA (Invitrogen)
  • Trypsin inhibitor buffer (see recipe)
  • Cryopreservation medium
  • Liquid nitrogen
  • 5% serum airway medium (see recipe)
  • Ussing chamber culture medium (see recipe)
  • 10‐cm or 100‐mm dishes
  • 12‐mm Millicell‐HA culture plate inserts (Millipore)
  • 2‐ml cryogenic vials
  • 24‐well tissue culture plates
  • Additional reagents and equipment for culturing of mammalian cells and counting viable cells by trypan blue exclusion ( appendix 3G) and generating airway epithelial cells (see protocol 1)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 4: Gene Transfer to Polarized Airway Epithelia

  Materials
  • Hanks’ balanced salt solution (HBSS; appendix 2D)
  • 25% and 40% (w/v) sucrose in HBSS
  • Lactose storage buffer (see recipe)
  • 8 mg/ml polybrene in H 2O (store in aliquots at −20°C)
  • Ussing chamber culture medium (see recipe)
  • Polarized airway epithelial cells grown on Millicell‐HA culture plate inserts (see protocol 3)
  • Ham's F‐12 medium (Life Technologies)
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 0.45‐µm filter (Millipore)
  • Sorvall RC‐26 Plus centrifuge with SS‐34 rotor (or equivalent) and centrifuge tubes accommodating 40 ml
  • Beckman ultracentrifuge with SW 41 rotor (or equivalent) and SW 41 centrifuge tubes
  • Filtron 100K concentrator (PALL)
  • 58°C water bath
  • 12,000‐ to 14,000‐Da MWCO dialysis membrane (Life Technologies)
  • Additional reagents and equipment for generating and/or purifying recombinant retrovirus and lentivirus (units 12.5& 12.10), adeno‐associated virus (unit 12.9), or adenovirus (unit 12.4)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 5: Generation of Human Tracheal Xenografts

  Materials
  • Fisher 344 rats, 200 to 250 g, male (Harlan Bioproducts for Science or Charles River Labs
  • CO 2 (for rat euthanization)
  • 70% (v/v) ethanol
  • Ice
  • MEM medium (Life Technologies), 4°C
  • Primary airway epithelial cells (see protocol 1) at ∼80% confluency
  • Medium C (see recipe)
  • nu/nu athymic mice, 20 to 25 g, male (Harlan Bioproducts for Science)
  • Ketamine
  • Xylazine
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Povidone‐iodine
  • Ham's F‐12 medium (Life Technologies)
  • Silastic tubing (0.030 in. i.d. × 0.065 in. o.d.; Dow Corning)
  • Teflon tubing (0.031 in. i.d. × 0.063 in. o.d.; Thomas)
  • Adapter (0.8‐mm barb‐to‐barb connector; Bio‐Rad)
  • 0.035 in. diameter Chromel A steel wire (Hoskins Mfg.)
  • Small forceps (two per mouse) and sharp scissors (one per mouse)
  • 100‐mm tissue culture plates
  • Gas sterilization pouch (M.D. Industries) and gas sterilization apparatus
  • Styrofoam bed
  • 2‐ml screw‐cap tubes (Sarstedt)
  • −80°C freezer
  • 2–0 braided silk suture (e.g., Ethicon)
  • 20‐µl micropipettors
  • Hemostat
  • Small airtight transfer chamber to equilibrate xenograft cassettes in 5% CO 2
  • Sterile surgical drapes for mouse surgery
  • Self‐sealable autoclave sterilization pouch, (M.D. Industries)
  • Disposable skin stapler 35R (American Cyanamid)
  • Sterile cages for mice
  • 21‐G × 0.75‐in. Surflo winged infusion set (Terumo Medical)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 2: Generation of Newborn Ferret Tracheal Xenografts

  Materials
  • Newborn ferrets, 1 to 15 days old (Marshall Farms)
  • Pentobarbital
  • 70% (v/v) ethanol
  • Medium D (see recipe)
  • Ice
  • MEM medium (Life Technologies), 4°C
  • nu/nu athymic mice, 20 to 25 g, male (Harlan Bioproducts for Science)
  • 1‐ml syringes equipped with 25‐G needles
  • 15‐ml conical tubes
  • Rotator
  • 2–0 braided silk suture (e.g., Ethicon)
  • 0.035 in. diameter Chromel A steel wire (Hoskins Mfg.)
  • Hemostat
  • 100‐mm tissue culture plates
  • Small airtight transfer chamber to equilibrate xenograft cassettes in 5% CO 2
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 6: Gene Transfer to Human Tracheal Xenografts

  Materials
  • Primary proliferating human airway cells (for ex vivo transduction with retrovirus; see protocol 1) or mouse with fully differentiated xenograft (for in vivo transduction with adenovirus; see protocol 6)
  • Retroviral supernatant (unit 12.5) or purified recombinant adenovirus (unit 12.4)
  • Ham's F‐12 medium (Life Technologies)
  • 5 mM EGTA, sterile filtered
  • Additional reagents and equipment for transduction of proliferating human airway cells (see protocol 2) and generation/maintenance of xenografts (see protocol 6)

Basic Protocol 7: In Vivo Gene Delivery to the Lung

  Materials
  • Cotton rats (80–120 g; Virion Systems)
  • Isoflurane
  • Purified recombinant adenovirus (unit 12.4)
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Pentobarbital
  • 100% optimal cutting temperature (OCT) medium (Baxter) and 50% (v/v) OCT medium in PBS
  • Pulverized dry ice/isopentane slurry
  • Gauze pads
  • 50‐ml conical centrifuge tubes
  • Isopropyl alcohol swabs
  • Scalpel, forceps (two), and small sharp scissors
  • Disposable skin stapler 35R (American Cyanamid)
  • 1‐ml syringe with 30‐G needle
  • 18‐G AngioCath (Becton Dickinson) and 3‐ml syringe
  • Plastic embedding blocks (Baxter)
  • Cryostat

Support Protocol 1: Harvesting of Human Tracheal Xenografts for Morphologic Analysis to Evaluate Transgene Expression

  Materials
  • Mice harboring xenografts (see Basic Protocols protocol 65 and protocol 86)
  • CO 2
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Optimal cutting temperature (OCT) medium (Baxter)
  • Pulverized dry ice/isopentane slurry
  • Dissecting equipment including:
    • Small sharp scissors
    • Forceps
    • Razor blades
  • Parafilm
  • Kimwipes
  • Plastic embedding block (Baxter)
  • Cryostat

Support Protocol 2: Histochemical Detection of β‐Galactosidase Transgene Activity

  Materials
  • 6‐µm frozen sections of tracheal xenograft (see protocol 10)
  • 0.5% (v/v) glutaraldehyde in PBS (prepare fresh)
  • 1 mM MgCl 2 in PBS
  • 1% (v/v) glutaraldehyde in buffered formalin (prepare fresh)
  • Xgal staining solution (see recipe)
  • Optimal cutting temperature medium (OCT; Baxter)
  • Pulverized dry ice/isopentane slurry
  • Hematoxylin or neutral red stain

Support Protocol 3: Immunohistochemical Detection of β‐Galactosidase Transgene Activity

  Materials
  • 6‐µm frozen sections of tracheal xenograft (see protocol 10)
  • 4% (w/v) paraformaldehyde in PBS, pH 7.4 to 7.6, 4°C
  • 20% (v/v) goat serum in PBS, filtered through 0.45‐µm filter (prepare fresh)
  • 1.5% (v/v) goat serum/PBS containing 25 µg/ml chicken anti–β‐galactosidase antibody (purchase from Abcam), freshly prepared
  • Fluorescein isothiocyanate (FITC)–labeled goat anti‐chicken antibody (or other fluorescently labeled goat anti‐chicken secondary antibody)
  • Citifluor antifade mounting medium (Ted Pella)
  • Humidified (moist) chamber (unit 4.3)
  • Coverslips
  • Fluorescence microscope

Support Protocol 4: Fluorescent Detection of Green Fluorescent Protein to Evaluate Transgene Expression

  Materials
  • Tissue samples incorporating GFP reporter gene
  • 4% (w/v) paraformaldehyde in PBS, pH 7.4 to 7.6, 4°C
  • 10%, 20%, and 30% sucrose in PBS, 4°C
  • Phosphate‐buffered saline (PBS; appendix 2D), cold
  • Fluorescence microscope with 450‐ to 500‐nm FITC filter sets
  • Additional reagents and equipment for freezing and cryosectioning xenograft samples (see protocol 10)

Support Protocol 5: Histochemical Detection of Alkaline Phosphatase Transgene Activity

  Materials
  • 6‐µm frozen sections of tracheal xenograft (see protocol 10)
  • 0.5% (v/v) glutaraldehyde in PBS (prepare fresh)
  • 1 mM MgCl 2 in PBS
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Alkaline phosphatase prestaining buffer (see recipe)
  • Alkaline phosphatase staining solution (see recipe)
  • Neutral red stain
  • Aqueous mounting medium
  • 65°C waterbath or oven
  • Coverslips
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Figures

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Literature Cited

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