Preparation of Adenovirus‐Polylysine‐DNA Complexes

Matt Cotten1, Adam Baker1, Max L. Birnstiel1, Kurt Zatloukal2, Ernst Wagner3

1 Institute for Molecular Pathology, Vienna, 2 University of Graz Medical School, Graz, 3 Boehringer Ingelheim R & D Vienna, Vienna
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.3
DOI:  10.1002/0471142905.hg1203s11
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes preparation of adenovirus‐polylysine‐DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin‐polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols descibes preparation of streptavidin‐polylysine and transferrin‐polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine‐virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine‐modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Biotinylated Adenovirus and Streptavidin‐Polylysine
  • Alternate Protocol 1: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Polylysine‐Adenovirus
  • Alternate Protocol 2: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Free Virus
  • Support Protocol 1: Growth and Purification of Adenovirus
  • Support Protocol 2: Biotinylation of Adenovirus
  • Support Protocol 3: Psoralen Inactivation of Adenovirus
  • Quantitation of Adenovirus
  • Support Protocol 4: Quantitation of Adenoviral Particles by Bradford Protein Assay
  • Support Protocol 5: Quantitation of Adenoviral Particles by Cytopathic Effect Assay
  • Support Protocol 6: Preparation of Streptavidin‐Polylysine
  • Preparation of LPS‐Free DNA
  • Support Protocol 7: Removing LPS by Triton X‐114 Extraction
  • Support Protocol 8: Removing LPS by Polymyxin B–Affinity Chromatography
  • Support Protocol 9: Preparation of Transferrin‐Polylysine Conjugates
  • Support Protocol 10: Preparation of Polylysine‐Modified Adenovirus Using Transglutaminase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Biotinylated Adenovirus and Streptavidin‐Polylysine

  Materials
  • Biotinylated adenovirus (see protocol 5), psoralen‐inactivated (see protocol 6; optional)
  • recipeHEPES‐buffered saline, pH 7.4 (HBS; see recipe)
  • Streptavidin‐polylysine (see protocol 9)
  • Lipopolysaccharide (LPS)‐free DNA (see protocol 10 or protocol 118)
  • Transferrin‐polylysine (see protocol 12, or Boehringer Ingelheim Bioproducts)
  • Cells to be transfected, <80% confluent in 24‐well tissue culture plate
  • Complete culture medium appropriate for the cells to be transfected with reduced serum (e.g., 2% [v/v] horse serum [HS] or 2% FBS)
  • Complete culture medium appropriate for the cells to be transfected with normal serum concentration
  • Additional reagents and equipment for tissue culture ( appendix 3G) and analysis for gene expression (e.g., CPMB UNITS )

Alternate Protocol 1: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Polylysine‐Adenovirus

  • 0.5 to 1 × 1012 particles/ml polylysine‐adenovirus (see protocol 13)
  • 1 mg/ml polylysine‐L‐lysine hydrobromide (30,000 to 70,000 mol. wt., Sigma) or transferrin‐polylysine containing 0.4 µg poly‐L‐lysine (see protocol 11)
  • Additional reagents and equipment for tissue culture ( appendix 3G)

Alternate Protocol 2: Preparation of Adenovirus‐Polylysine‐DNA Transfection Complexes Using Free Virus

  • Poly‐L‐lysine hydrobromide (70,000 to 150,000 mol. wt., Sigma) or transferrin‐polylysine conjugate (see protocol 11)
  • CsCl‐purified adenovirus (see protocol 4; may be psoralen inactivated, see protocol 6), at >0.1 × 1012 particles/ml
  • Cells to be transfected, at 20,000 to 50,000 cells/well in 24‐well tissue culture plate
  • recipeBuffered complete Dulbecco's minimal essential medium supplemented with 2% (v/v) horse serum (DMEM/2% HS; see recipe)
  • recipeBuffered complete DMEM supplemented with 10% (v/v) FBS (DMEM/10% FBS; see recipe)
  • Additional reagents and equipment for tissue culture ( appendix 3G) and assaying for gene expression (e.g., CPMB UNITS )

Support Protocol 1: Growth and Purification of Adenovirus

  Materials
  • Cells: 293 (ATCC CRL‐1573) or W162 (Weinberg and Ketner, ), 80% confluent in 180‐cm2 flask
  • recipeBuffered complete minimal essential medium alpha supplemented with 10% (v/v) newborn calf serum (MEMα/10% NCS; see recipe)
  • recipeBuffered complete Dulbecco's modified Eagle medium (DMEM) supplemented with 2% (v/v) horse serum (DMEM/2% HS; see recipe)
  • recipeBuffered complete DMEM supplemented with 10% FBS (DMEM/10% FBS; see recipe)
  • Adenovirus stock: e.g., Ad5 dl312 (Jones and Shenk, ) or Ad5 dl1014 (Bridge and Ketner, )
  • 20 mM N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), pH 7.4
  • recipe100 mM phenylmethylsulfonyl fluoride (PMSF) in ethanol (see recipe)
  • 1,1,2‐trichlorotrifluoroethane (freon; EM Science)
  • recipeHEPES‐buffered saline, pH 7.4 (HBS; see recipe)
  • recipeHBS/40% (v/v) glycerol (see recipe)
  • recipeCsCl solutions: 1.31 g/cm3 and 1.64 g/cm3, pH 7.4 (see recipe)
  • Liquid nitrogen or dry ice/ethanol bath
  • 180‐cm2 tissue culture flasks
  • 50‐ml centrifuge tubes or 250‐ml centrifuge bottles
  • Heraeus centrifuge and 2705 rotor or Sorvall centrifuge and GSA rotor or equivalent
  • 360‐W sonicating water bath
  • Long hypodermic needles:
    • 0.9 (20‐G) × 120–mm needle (Althin Mediplast AB) for VTi 65 tube
    • 21‐G × 3‐⅛‐in. (79.4‐cm) needle (Erosa, Rose GmBH) for VTi 50 tube
    • VTi 50 and VTi 65 centrifuge tubes (Beckman)
  • Ultracentrifuge and VTi 50 and VTi 65 rotors or equivalent
  • 5‐ml syringe and 21‐G needles
  • Refractometer
  • Additional reagents and equipment for tissue culture ( appendix 3G) and quantitating virus by measuring protein content (see protocol 7)
CAUTION: Cell culture and virus preparation should be carried out in a cell culture facility with containment at a level appropriate for the virus used (see Chapter 12 Introduction or consult local safety authorities).

Support Protocol 2: Biotinylation of Adenovirus

  Materials
  • Sulfosuccinimidyl‐6‐(biotinimido)hexamate (NHS‐LC‐biotin, Pierce; store desiccated)
  • 5 mM N‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), pH 7.9
  • Purified adenovirus at 0.5 to 3 × 1012 particles/ml, direct from CsCl gradient, in recipeHEPES‐buffered saline (HBS) or in recipeHBS/40% glycerol (see protocol 4)
  • recipeHBS/40% (v/v) glycerol (see recipe)
  • Sephadex G‐50 Nick column or Sephadex G‐25 PD‐10 column (Pharmacia Biotech)
  • Additional reagents and equipment for dialysis (e.g., CPMB APPENDIX )

Support Protocol 3: Psoralen Inactivation of Adenovirus

  Materials
  • Purified adenovirus in recipeHEPES‐buffered saline (HBS), recipeHBS/40% glycerol, or in CsCl direct from gradient (see protocol 4) at >0.1 × 1012 particles/ml
  • recipe33 mg/ml 8‐methoxypsoralen in DMSO (see recipe)
  • 4‐ or 24‐well tissue culture plate (e.g., Nunc)
  • 360‐nm (long‐wavelength) UV light source: e.g., model TL‐33 with 6 × 15‐W bulbs producing 12,000 to 13,000 µW/cm2 at 3‐cm distance (UVP)
  • Sephadex G‐25 PD‐10 or Sephadex G‐50 Nick gel filtration column (Pharmacia Biotech), equilibrated with recipeHBS/40% (v/v) glycerol
  • Additional reagents and equipment for quantitation of adenovirus by protein concentration (see protocol 7)

Support Protocol 4: Quantitation of Adenoviral Particles by Bradford Protein Assay

  Materials
  • Purified virus sample (see protocol 4) or biotinylated (see protocol 5) or psoralen‐inactivated (see protocol 6) virus
  • Bradford reagent (Bio‐Rad)
  • Protein standard (e.g., 1 mg/ml fraction V BSA, Boehringer Mannheim)
  • Purified virus standard
  • Disposable plastic cuvettes (Sarstedt)

Support Protocol 5: Quantitation of Adenoviral Particles by Cytopathic Effect Assay

  Materials
  • Target cells susceptible to the strain of test adenovirus
  • Complete culture medium appropriate for target cells, e.g., recipeMEMα/10% NCS or recipeDMEM/10% FBS (see reciperecipes)
  • Purified test adenovirus (see protocol 4) at ∼1 × 1012 particles/ml
  • recipeBuffered complete Dulbecco's minimal essential medium supplemented with 2% (v/v) horse serum (DMEM/2% HS; see recipe)
  • PBS ( appendix 2D)
  • 4% (w/v) formaldehyde/150 mM NaCl
  • 0.1% (w/v) crystal violet/2% (v/v) ethanol
  • recipeCrystal violet eluting solution (see recipe; optional)
  • 24‐well tissue culture plate
  • Additional reagents and equipment for tissue culture ( appendix 3G)

Support Protocol 6: Preparation of Streptavidin‐Polylysine

  • Streptavidin (Pierce)
  • Sephadex G‐25 (Pharmacia Biotech)
  • recipeHEPES‐buffered saline, pH 7.4 (HBS; see recipe)
  • N‐succinimidyl‐3‐(2‐pyridyldithiol)propionate (SPDP; Pharmacia Biotech)
  • 60% and 100% ethanol
  • Dithiothreitol (DTT) solution: 10 mg DTT/0.6 ml recipeHBS
  • 100 mg/ml gel‐filtered polylysine solution (see protocol 12, steps to )
  • 0.1 M and 2 M N‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), degassed
  • 0.25 M NaCl, degassed
  • Sephadex G‐25 Superfine resin (Pharmacia Biotech)
  • Poly‐L‐lysine hydrobromide (70,000 to 150,000 mol. wt., Sigma)
  • recipeNinhydrin reagents A, B, and C (see recipe)
  • Dithiothreitol (DTT)
  • Ellman reagent solution: 4 mg/ml Ellman reagent (EM Science)/0.1 M HEPES
  • 0.5 M NaCl
  • Macro‐Prep high S resin (Bio‐Rad)
  • 0.5 M NaCl/20 mM HEPES
  • 3 M NaCl/20 mM HEPES
  • 10 × 100–mm and 10 × 300–mm liquid chromatography columns (HR 10/10 and HR 10/30, Pharmacia Biotech)
  • 12 × 75– or 17 × 100–mm polypropylene tubes with caps, sterile
  • Liquid chromatography separation system: HPLC, FPLC, or equivalent system with gradient maker, UV detector, and fraction collector for gel‐filtration and ion‐exchange chromatography
  • Microdialysis system (Life Technologies) with MWCO 6000 to 8000 membrane (Life Technologies)
  • Argon or nitrogen supply (for the exclusion of oxygen)
  • Additional reagents and equipment for gel‐filtration chromatography (e.g., CPMB UNIT ), dialysis (e.g., CPMB APPENDIX ) and ion‐exchange chromatography (e.g., CPMB UNIT )
CAUTION: Several of the chemical substances listed—e.g., phenol, potassium cyanide, pyridine, and ninhydrin—are hazardous and require special handling.

Support Protocol 7: Removing LPS by Triton X‐114 Extraction

  Materials
  • Overnight culture of plasmid‐transformed bacteria
  • TE buffer, pH 7.4 ( appendix 2D)
  • recipeTriton X‐114, preequilibrated (see recipe)
  • 3 M sodium acetate, pH 7.5
  • Isopropanol
  • 80% ethanol, −20°C
  • Additional reagents and equipment for plasmid DNA purification (unit 5.3 & CPMB UNITS & ) and quantitation of DNA ( appendix 3D)

Support Protocol 8: Removing LPS by Polymyxin B–Affinity Chromatography

  Materials
  • Plasmid DNA (see protocol 10, steps to )
  • TE buffer, pH 7.4 to 7.5 ( appendix 2D), LPS‐free
  • Polymyxin B resin (Affi‐Prep polymyxin, Bio‐Rad)
  • 0.1 and 0.3 N NaOH, LPS‐free
  • 3 M sodium acetate, pH 7.5, LPS‐free
  • 100% and 80% ethanol, −20°C, LPS‐free
  • pH paper
  • Disposable columns (e.g., Poly‐prep, Bio‐Rad)
  • Additional reagents and equipment for quantitation of DNA ( appendix 3D)
NOTE: LPS‐free glassware, plasticware, and reagents should be used for handling the DNA after polymyxin B chromatography.

Support Protocol 9: Preparation of Transferrin‐Polylysine Conjugates

  Materials
  • Poly‐L‐lysine hydrobromide (mol. wt. 30,000 to 70,000, equal to an average degree of polymerization between 100 and 400 lysine monomers; Sigma)
  • 0.25 and 0.5 M NaCl
  • Sephadex G‐25 Superfine resin (Pharmacia Biotech)
  • Transferrin (human, iron‐free; Sigma or Biotest)
  • 30 mM sodium acetate buffer, adjust to pH 5.0 with acetic acid
  • 100 mM sodium periodate solution: 21.4 mg/ml sodium periodate/30 mM sodium acetate buffer, pH 5.0
  • 2 M N‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES), pH 7.9
  • Sodium cyanoborohydride (EM Science)
  • Macro‐Prep high S resin (Bio‐Rad)
  • recipeHEPES‐buffered saline, pH 7.4 (HBS; see recipe)
  • recipe10 mM ferric citrate buffer, pH 7.5 to 8.0 (see recipe)
  • 10 × 100–mm and 10 × 300–mm liquid chromatography columns (e.g., HR 10/10 and HR 10/30, Pharmacia Biotech)
  • Liquid chromatography separation system: HPLC, FPLC, or equivalent system with gradient‐former, UV detector, and fraction collector for gel‐filtration and ion‐exchange chromatography
  • 12 × 75– or 17 × 100–mm polypropylene tubes with caps, sterile
  • Argon or nitrogen supply (for exclusion of oxygen)
  • Microdialysis system (Life Technologies) with MWCO 6000 to 8000 membrane (Life Technologies)
  • Additional reagents and equipment for gel‐filtration liquid chromatography (e.g., CPMB UNIT ), ion exchange chromatography (e.g., CPMB UNIT ), ninhydrin assay (see protocol 9, steps and ), and dialysis (e.g., CPMB APPENDIX )
CAUTION: Several of the chemical substances listed—e.g., sodium cyanoborohydride and 96% acetic acid—are hazardous and require special handling.

Support Protocol 10: Preparation of Polylysine‐Modified Adenovirus Using Transglutaminase

  Materials
  • Adenovirus (see protocol 4), active or psoralen‐inactivated (see protocol 6)
  • recipeTransglutaminase reaction buffer (see recipe)
  • recipe8 µM transglutaminase (see recipe)
  • 10 mg/ml poly‐L‐lysine hydrobromide (30,000 to 70,000 mol. wt., Sigma)
  • 1 M CaCl 2
  • 0.5 M EDTA ( appendix 2D)
  • recipeCsCl solutions (see recipe)
  • Glycerol
  • Ultracentrifuge
  • SW 41 rotor (Beckman) and centrifuge tubes
  • VTi 65 rotor (Beckman) and centrifuge tubes
  • Refractometer
  • Additional reagents and equipment for gel filtration chromatography (CPMB UNIT ) and determination of virus titer based on protein concentration (see protocol 7)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aida, Y. and Pabst, M.J. 1990. Removal of endotoxin from protein solutions by phase separation using Triton X‐114. J. Immunol. Methods 132:191‐195.
   Arscott, P.G., Li, A.Z., and Bloomfield, V.A. 1990. Condensation of DNA by trivalent cations. 1. Effects of DNA length and topology on the size and shape of condensed particles. Biopolymers 30:619‐630.
   Batra, R.K., Wang‐Johanning, F., Wagner, E., Garver, R.I. Jr., and Curiel, D.T. 1994. Receptor‐mediated gene delivery employing lectin‐binding specificity. Gene Ther. 1:255‐260.
   Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X‐114 solution. J. Biol. Chem. 256:1604‐1607.
   Böttger, M., Vogel, F., Platzer, M., Kiessling, U., Grade, K., and Strauss, M. 1988. Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection. Biochim. Biophys. Acta. 950:221‐228.
   Bridge, E. and Ketner, G. 1989. Redundant control of adenovirus late gene expression by early region 4. J. Virol. 63:631‐638.
   Buschle, M., Cotten, M., Kirlappos, H., Mechtler, K., Schaffner, G., Zauner, W., Birnstiel, M.L., and Wagner, E. 1995. Receptor‐mediated gene transfer into human T‐lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex. Hum. Gene Ther. 6:753‐761.
   Carrasco, L. 1994. Entry of animal viruses and macromolecules into cells. FEBS Lett. 350:151‐154.
   Carrasco, L. 1995. Modification of membrane permeability by animal viruses. Adv. Virus Res. 45:61‐112.
   Chardonnet, Y. and Dales, S. 1970. Early events in the interaction of adenoviruses with HeLa cells: Penetration of type 5 and intracellular release of the DNA genome. Virology. 40:462‐477.
   Chattoraj, D.K., Gosule, L.C., and Schellman, J.A. 1978. DNA condensation with polyamines. J. Mol. Biol. 121:327‐337.
   Chen, P., Ornelles, D., and Shenk, T. 1993. The adenovirus L3 23‐kilodalton proteinase cleaves the amino‐terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells. J. Virol. 67:3507‐3514.
   Chiocca, S., Kurzbauer, R., Schaffner, G., Baker, A., Mautner, V., and Cotten, M. 1996. The complete DNA sequence and genomic organization of the avian adenovirus CELO. J. Virol. 70:2939‐2949.
   Chiou, H.C., Tangco, M.V., Levine, S.M., Robertson, D., Kormis, K., Wu, C.H., and Wu, G.Y. 1994. Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein‐polylysine carriers. Nucl. Acids Res. 24:5439‐5446.
   Cotten, M. 1995. The entry mechanism of adenovirus and some solutions to the toxicity problems associated with adenovirus‐augmented, receptor‐mediated gene delivery. Curr. Top. Microbiol. Immunol. 199:283‐295.
   Cotten, M. and Wagner, E. 1993. Nonviral approaches to gene therapy. Curr. Opin. Biotechnol. 4:705‐710.
   Cotten, M. and Weber, J.M. 1995. The adenovirus protease is required for virus entry into host cells. Virology. 213:494‐502.
   Cotten, M., Wagner, E., Zatloukal, K., Phillips, S., Curiel, D., and Birnstiel, M.L. 1992. High‐efficiency receptor‐mediated delivery of small and large (48‐kb) gene constructs using the endosome‐disruption activity of defective or chemically‐inactivated adenovirus particles. Proc. Natl. Acad. Sci. U.S.A. 89:6094‐6098.
   Cotten, M., Wagner, E., Zatloukal, K., and Birnstiel, M.L. 1993. Chicken adenovirus (CELO virus) particles augment receptor‐mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants. J. Virol. 67:3777‐3785.
   Cotten, M., Saltik, M., Kursa, M., Wagner, E., Maass, G., and Birnstiel, M. 1994a. Psoralen treatment of adenovirus particles eliminates virus replication and transcription while maintaining the endosomolytic activity of the virus capsid. Virology. 205:254‐261.
   Cotten, M., Baker, A., Saltik, M., Wagner, E., and Buschle, M. 1994b. Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus. Gene Ther. 1:239‐246.
   Cristiano, R.J., Smith, L.C., Kay, M.A., Brinkley, B.R., and Woo, S.L.C. 1993. Hepatic gene therapy: Efficient gene delivery and expression in primary hepatocytes utilizing a conjugated denovirus‐DNA complex. Proc. Natl. Acad. Sci. U.S.A. 90:11548‐11552.
   Curiel, D.T. 1993. Adenovirus facilitation of molecular conjugate–mediated gene transfer. Prog. Med. Virol. 40:1‐18.
   Curiel, D.T., Agarwal, S., Wagner, E., and Cotten, M. 1991. Adenovirus enhancement of transferrin‐polylysine mediated gene delivery. Proc. Natl. Acad. Sci. U.S.A. 88:8850‐8854.
   Curiel, D., Wagner, E., Cotten, M., Birnstiel, M.L., Li, C., Loechel, S., Agarwal, S., and Hu, P. 1992. High‐efficiency gene transfer mediated by adenovirus coupled to DNA‐polylysine complexes via an antibody bridge. Hum. Gene Ther. 3:147‐154.
   Defer, C., Belin, M., Caillet‐Boudin, M., and Boulanger, P. 1990. Human adenovirus‐host cell interactions: Comparative study with members of subgroups B and C. J. Virol. 64:3661‐3673.
   Douglas, J.T. and Curiel, D.T. 1995. Targeted gene therapy. Tumor Targeting. 1:67‐84.
   Fernández‐Puentes, C. and Carrasco, L. 1980. Viral infection permeabilizes mammalian cells to protein toxins. Cell. 20:769‐775.
   Findeis, M.A., Wu, C.H., and Wu, G.Y. 1994. Ligand‐based carrier systems for delivery of DNA to hepatocytes. Methods Enzymol. 247:341‐351.
   Fisher, K.J. and Wilson, J.M. 1994. Biochemical and functional analysis of an adenovirus‐based ligand complex for gene transfer. Biochem. J. 299:49‐58.
   Fitzgerald, D., Padmanabhan, R., Pastan, I., and Willingham, M. 1983. Adenovirus‐induced release of epidermal growth factor and Pseudomonas toxin into the cytosol of KB cells during receptor‐mediated endocytosis. Cell. 32:607‐617.
   Goldsmith, K.T., Curiel, D.T., Engler, J.A., and Garver, R.I. Jr. 1994. Trans complementation of an E1A‐deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells. Hum. Gene Ther. 5:1341‐8
   Greber, U.F., Willetts, M., Webster, P., and Helenius, A. 1993. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 75:1‐20.
   Griffith, O.M. 1979. Techniques of Preparative, Zonal and Continuous Flow Ultracentrifugation. Beckman Instruments, Palo Alto, Calif.
   Jones, N. and Shenk, T. 1979. An adenovirus type 5 early gene function regulates expression of other early genes. Proc. Natl. Acad. Sci. U.S.A. 76:3665‐3669.
   Kaneda, Y., Iwai, K., and Uchida, T. 1989. Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science. 243:375‐378.
   Ledley, F. 1994. Nonviral gene therapy. Curr. Opin. Biotechnol. 5:626‐636.
   Lemay, P., Boudin, M., Milleville, M., and Boulanger, P. 1980. Human adenovirus type 2 protein IIIa. I. Purification and characterization. Virology. 101:131‐143.
   Manthorpe, M., Cornefert‐Jensen, F., Hartikka, J., Felgner, J., Rundell, A., Margalith, M., and Dwarki, V. 1993. Gene therapy by intramuscular injection of plasmid DNA: Studies on firefly luciferase gene expression in mice. Hum. Gene Ther. 4:419‐431.
   Perales, J.C., Ferkol, T., Molas, M., and Hanson, R.W. 1994. An evaluation of receptor‐mediated gene transfer using synthetic DNA‐ligand complexes. Eur. J. Biochem. 226:255‐266.
   Scaria, A., Curiel, D.T., and Kay, M.A. 1995. Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus‐polylysine‐DNA complexes. Gene Ther. 2:295‐298.
   Schreiber, M., Baumann, B., Cotten, M., Angel, P., and Wagner, E.F. 1995. c‐Fos is an essential component of the mammalian UV response. EMBO J. 14:5338‐5349.
   Thurnher, M., Wagner, E., Clausen, H., Mechtler, K., Rusconi, S., Dinter, A., Berger, E., Birnstiel, M., and Cotten, M. 1994. Carbohydrate receptor–mediated gene transfer to human T‐leukemic cells. Glycobiology. 4:429‐435.
   Tomita, N., Higaki, J., Morishita, R., Kato, K., Mikami, H., Kaneda, Y., and Ogihara, T. 1992. Direct in vivo gene introduction into rat kidney. Biochem. Biophys. Res. Commun. 186:129‐134.
   von Rüden, T., Stingl, L., Cotten, M., Wagner, E., and Zatloukal, K. 1995. Generation of high titer retroviral vectors following receptor‐mediated adenovirus‐augmented transfection of packaging cell lines. BioTechniques. 18:484‐489.
   Wagner, E., Cotten, M., Mechtler, K., Kirlappos, H., and Birnstiel, M.L. 1991a. DNA‐binding transferrin conjugates as functional gene‐delivery agents: Synthesis by linkage of polylysine or ethidium homodimer to transferrin carbohydrate moiety. Bioconjugate Chem. 2:226‐231.
   Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M.L. 1991b. Transferrin‐polycation‐DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. U.S.A. 88:4255‐4259.
   Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K., Curiel, D., and Birnstiel, M.L. 1992. Coupling of adenovirus to transferrin‐polylysine‐DNA complexes greatly enhances receptor‐mediated gene delivery and expression of transfected cells. Proc. Natl. Acad. Sci. U.S.A. 89:6099‐6103.
   Wagner, E., Curiel, D., and Cotten, M. 1994. Delivery of drugs, proteins, and genes into cells using transferrin as a ligand for receptor‐mediated endocytosis. Adv. Drug Delivery Reviews. 14:113‐135.
   Weinberg, D.H. and Ketner, G. 1983. A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc. Natl. Acad. Sci. U.S.A. 80:5383‐5386.
   Wu, G., Zhan, P., Sze, L., Rosenberg, A., and Wu, C. 1994. Incorporation of adenovirus into a ligand‐based DNA carrier system results in retention of original receptor specificity and enhances targeted gene expression. J. Biol. Chem. 269:11542‐11546.
   Zatloukal, K., Wagner, E., Cotten, M., Phillips, S., Plank, C., Steinlein, P., Curiel, D., and Birnstiel, M.L. 1992. Transferrinfection: A highly efficient way to express gene constructs in eukaryotic cells, Ann. N.Y. Acad. Sci. 660:136‐153.
   Zatloukal, K., Cotten, M., Berger, M., Schmidt, W., Wagner, E., and Birnstiel, M.L. 1994. In vivo production of human factor VIII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor‐mediated adenovirus‐augmented gene delivery. Proc. Natl. Acad. Sci. U.S.A. 91:5148‐5152.
   Zatloukal, K., Schneeberger, A., Berger, M., Schmidt, W., Koszik, F., Kutil, R., Cotten, M., Wagner, E., Buschle, M., Maass, G., Payer, E., Stingl, G., and Birnstiel, M.L. 1995. Elicitation of a systemic and protective anti‐melanoma immune response by an IL‐2‐based vaccine: Assessment of critical cellular and molecular parameters. J. Immunol. 154:3406‐3419.
   Zauner, W., Blaas, D., Kücchler, E., and Wagner, E. 1995. Rhinovirus‐mediated endosomal release of transfection complexes. J. Virol. 69:1085‐1092.
   Zhang, Y. and Schneider, R.J. 1994. Adenovirus inhibition of cell translation facilitates release of virus particles and enhances degradation of the cytokeratin network. J. Virol. 68:2544‐2555.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library