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Gene Delivery to the Airway

Dongsheng Duan1,  Yulong Zhang1,  John F. Engelhardt1

1University of Iowa, Iowa City, Iowa

Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 13.9
DOI: 
10.1002/0471142905.hg1309s23
Online Posting Date: 
May, 2001
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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 human bronchial 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.

     
 
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Table of Contents

  • Unit 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 Airway Epithelial Monolayers
  • Basic Protocol 4: Gene Transfer to Polarized Airway Epithelia
  • Basic Protocol 5: Generation of Human Bronchial Xenografts
  • Basic Protocol 6: Gene Transfer to Human Bronchial Xenografts
  • Basic Protocol 7: In Vivo Gene Delivery to the Lung
  • Support Protocol 1: Harvesting of Human Bronchial Xenografts for Morphologic Analysis to Evaluate Transgene Expression
  • Support Protocol 2: Histochemical Detection of -Galactosidase Transgene Activity
  • Support Protocol 3: Immunohistochemical Detection of -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)
  • Media A, B, and C (see recipes)
  • 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
  • Dissecting equipment including forceps, scalpel, and 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% CO2 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 H2O (store in aliquots at –20°C)
  • Primary airway epithelial cells (see Basic 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 filter 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 vectors (unit 12.5) or producing and purifying adeno-associated viral vectors (unit 12.9)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 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 Airway Epithelial Monolayers

 Materials
  • Glacial acetic acid
  • Collagen, Type VI, acid-soluble, from human placenta (Sigma)
  • 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 Basic Protocol 1)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 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 H2O (store in aliquots at –20°C)
  • Ussing chamber culture medium (see recipe)
  • Polarized airway epithelial cells grown on Millicell-HA culture plate inserts (see Basic 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 (unit 12.5), adeno-associated virus (unit 12.9), or adenovirus (unit 12.4)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 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 Bronchial Xenografts

 Materials
  • Fisher 344 rats, 200 to 250 g, male (Harlan Bioproducts for Science or Charles River Labs
  • 70% ethanol
  • MEM medium (Life Technologies), 4°C
  • Medium C (see recipe)
  • Primary airway epithelial cells (see Basic Protocol 1) at ~80% confluency
  • 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)
  • Gas sterilization pouch (M.D. Industries) and gas sterilization apparatus
  • 2-ml screw-cap tubes (Sarstedt)
  • Adapter (0.8-mm barb-to-barb connector; Bio-Rad)
  • 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% CO2
  • Sterile surgical drapes for mouse surgery
  • Self-sealable autoclave sterilization pouch, (M.D. Industries)
  • Small forceps (2 per mouse) and sharp scissors (1 per mouse)
  • 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.

Basic Protocol 6: Gene Transfer to Human Bronchial Xenografts

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

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)
  • 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 (2), 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 Bronchial Xenografts for Morphologic Analysis to Evaluate Transgene Expression

 Materials
  • Mice harboring xenografts (see Basic Protocols 5 and 6)
  • PBS (appendix 2D)
  • Optimal cutting temperature (OCT) medium (Baxter)
  • Pulverized dry ice/isopentane slurry
  • Dissecting equipment: small sharp scissors, forceps, and razor blades
  • Plastic embedding block (Baxter)
  • Cryostat

Support Protocol 2: Histochemical Detection of -Galactosidase Transgene Activity

 Materials
  • 6-µm frozen sections of bronchial xenograft (see Support Protocol 1)
  • 0.5% (v/v) glutaraldehyde in PBS (prepare fresh)
  • 1 mM MgCl2 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 -Galactosidase Transgene Activity

 Materials
  • 6-µm frozen sections of bronchial xenograft (see Support Protocol 1)
  • Methanol, –20°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 rabbit anti–-galactosidase antibody (purchase from 5 Prime3 Prime), freshly prepared
  • Fluorescein isothiocyanate (FITC)–labeled goat anti-rabbit antibody (or other fluorescently labeled goat anti-rabbit 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
  • 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 Support Protocol 1)

Support Protocol 5: Histochemical Detection of Alkaline Phosphatase Transgene Activity

 Materials
  • 6-µm frozen sections of bronchial xenograft (see Support Protocol 1)
  • 0.5% (v/v) glutaraldehyde in PBS (prepare fresh)
  • 1 mM MgCl2 in PBS
  • 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

  • Figure 13.9.1
    In vitro cultures of polarized airway epithelial cells. The monolayer of primary airway epithelial cells are grown on collagen-coated Millipore cell culture inserts (the total growth area is 0.6 cm2) placed in wells of a 24-well tissue culture plate. The support filter is made from mixed esters of cellulose nitrate and acetate and subsequently coated with liquid collagen. During culturing the basolateral sides of the human airway epithelia are exposed to Ussing chamber culture medium (~500 µl is added to the outside of each insert). The apical surfaces are exposed to the air. Scanning electron micrographs kindly provided by Zabner et al., 1996; Bar: right panels, 5 µm; left panels, 37.5 µm. Reproduced with permission from American Society for Microbiology.

    View Image
  • Figure 13.9.2
    Human bronchial xenograft model. Subcutaneous implants of xenograft human airways are generated from primary bronchial human airway cells transplanted onto denuded rat tracheas. Primary cells can be genetically manipulated ex vivo with vectors such as recombinant retrovirus and adeno-associated virus (see Basic Protocol 2). Alternatively, xenografts can be generated from genetically unaltered primary cells and gene-transfer studies performed in vivo following reconstitution of grafts. Typically, a fully differentiated mucociliary epithelium is obtained by 4 weeks post-transplantation. Identifiers on micrograph: B, basal cell; Ci, ciliated cell; I, intermediate cell; G, goblet cell. Bar = 20 µm).

    View Image
  • Figure 13.9.3
    Xenograft cassettes are generated from a series of defined types of tubing as defined in (A). Tube a, 2.5-cm Silastic tubing; tube b, 1.9-cm Silastic tubing; tube c, 4.5-cm Silastic tubing; tube d, 3.2-cm Teflon tubing; tube e, adapter. Tubing is connected to denuded rat tracheas by a series of suture ligations as shown in (B).

    View Image
  • Figure 13.9.4
    Transplantation of bronchial xenografts in nu/nu mice. (A) The mouse on the left represents a schematic view of subcutaneous transplantation of the xenograft cassette. (B) Four incisions are made as marked by arrows and the xenograft cassette guided subcutaneously using forceps, so that one port exits through the back of the neck and the other port through the main incision. Surgical staples are used to close incisions as marked by open arrowheads. (C)The right diagram shows the resultant xenografted mouse after 1 week post transplantation, when staples are removed. The staples marked by closed arrowheads in panel C are used to maintain the position of the cassette and prevent subcutaneous migration (typically it may be necessary to leave these staples in for 2 to 3 weeks).

    View Image

Literature Cited

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