Use of the Hemagglutinating Virus of Japan (HVJ) Envelope as a Versatile Delivery System for Nucleic Acids and Proteins to Leukocytes In Vitro

Yoshitaka Kondo1, Keizo Miyata1, Fuminori Kato1

1 Life Science Research Laboratory, Central Research Institute, Ishihara Sangyo Kaisha, Ltd., Kusatsu, Shiga, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.17D
DOI:  10.1002/0471142735.im1017ds89
Online Posting Date:  April, 2010
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Abstract

The hemagglutinating virus of Japan (HVJ; also called the Sendai virus) envelope has been developed as a safe and efficient non‐viral vector. Because replication and transcription of genomic RNA is inactivated by β‐propiolactone treatment or UV‐irradiation, the HVJ envelope is extremely safe. This unit describes the method of transfection of siRNA and protein with the HVJ envelope. Curr. Protoc. Immunol. 89:10.17D.1‐10.17D.9. © 2010 by John Wiley & Sons, Inc.

Keywords: hemagglutinating virus of Japan; Sendai virus; HVJ envelope; siRNA transfection

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transfection of siRNA
  • Basic Protocol 2: Transfection of Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transfection of siRNA

  Materials
  • HVJ envelope (see recipe; suspension containing ∼1 × 1011 particles/ml), ice cold
  • 0.1 to 0.5 µg/µl siRNA, ice cold
  • 2% Triton X‐100 (see recipe), ice cold
  • PBS (see recipe), ice cold
  • 10 mg/ml protamine sulfate (see recipe), ice cold
  • Suspension of cells in culture medium (0.4–2 × 106 cells/0.5 ml)
  • Appropriate culture medium
  • 2.0‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • 6‐well plates
  • 37°C, 5% CO 2 incubator
NOTE:Perform steps 1 through 6 on ice.

Basic Protocol 2: Transfection of Protein

  Materials
  • HVJ envelope (see recipe; suspension containing ∼1 × 1011 particles/ml), ice cold
  • 1 mg/ml protamine sulfate (see recipe), ice cold
  • 0.5 to 2 mg/ml protein or peptide, ice cold
  • 2% Triton X‐100 (see recipe), ice cold
  • PBS (see recipe), ice cold
  • Suspension of cells (0.4–2 × 106 cells/0.5 ml)
  • Appropriate culture medium
  • 2.0‐ml microcentrifuge tube
  • Refrigerated microcentrifuge
  • 6‐well plates
  • 37°C, 5% CO 2 incubator
NOTE:Steps 1 through 8 should be performed on ice.
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Figures

  •   FigureFigure 10.17.1 Mechanism of transfection of the HVJ envelope. Beta‐propiolactone treatment or UV irradiation destroys the genome of HVJ, and the resultant HVJ envelope retains fusion activity but cannot replicate. Detergent treatment makes the HVJ envelope highly permeable, enabling the HVJ envelope to incorporate nucleic acids (siRNA, plasmid DNA, synthetic oligo DNA) and proteins. These molecules are introduced into cells by fusion between the viral envelope and cell membrane.
  •   FigureFigure 10.17.2 Outline of transfection using the HVJ envelope.
  •   FigureFigure 10.17.3 Effects of Eg5 siRNA introduction using the HVJ envelope on the viability of Jurkat cells. Two days after transfection, viability of Jurkat cells was measured by WST‐1. Results are expressed as relative cell viability to non‐treatment control.
  •   FigureFigure 10.17.4 Cytotoxic effects of RNase T1 introduction using the HVJ envelope. RNase T1 was introduced into K562 cells using the HVJ envelope. Twenty‐four hours after transfection, cell survival was analyzed by WST‐8. Results are expressed as relative cell viability to non‐treatment control.

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Literature Cited

Literature Cited
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   Kaneda, Y., Nakajima, T., Nishikawa, T., Yamamoto, S., Ikegami, H., Suzuki, N., Nakamura, H., Morishita, R., and Kotani, H. 2002. Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system. Mol. Ther. 6:219‐226.
   Kaneda, Y., Yamamoto, S., and Nakajima, T. 2005. Development of HVJ envelope vector and its application to gene therapy. Adv. Genet. 53PA:307‐332.
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