Efficient Delivery of Antibody Into Living Cells Using Hemagglutinating Virus of Japan (HVJ) Envelope

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 2.16
DOI:  10.1002/0471142735.im0216s89
Online Posting Date:  April, 2010
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Abstract

This unit describes a novel method for antibody delivery into living cells using HVJ (hemagglutinating virus of Japan) envelope, an inactivated Sendai virus particle. Curr. Protoc. Immunol. 89:2.16.1‐2.16.12. © 2010 by John Wiley & Sons, Inc.

Keywords: intracellular delivery of antibody; hemagglutinating virus of Japan; HVJ; Sendai virus; nucleocapsid protein (NP); Z domain; Protein A; ZZ‐NP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Introduction of Antibody into Living Cells Using HVJ Envelope
  • Alternate Protocol 1: HVJ Envelope‐Mediated Intracellular Delivery of an Affinity‐Tagged Protein (Antibody)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Introduction of Antibody into Living Cells Using HVJ Envelope

  Materials
  • HVJ envelope, ∼1 × 1011 particles/ml (provided with GenomONE‐CAb kit, Ishihara Sangyo Kaisha Ltd., or see recipe in Reagents and Solutions), ice‐cold
  • 0.5 mg/ml ZZ‐NP, (provided with GenomONE‐CAb kit, Ishihara Sangyo Kaisha Ltd., or see recipe in Reagents and Solutions)
  • 2% (w/v) Triton X‐100 (see recipe), ice‐cold
  • 0.1 to 0.5 mg/ml of antibody to be delivered into cells of choice (IgG is recommended; see ; also see annotation to step 5), ice‐cold
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • 1 mg/ml protamine sulfate (see recipe), ice‐cold
  • Cells successfully tested: HAEC, HeLa S3, HeLa, HT1080, P19, SAS, NIH/3T3, A549, Hs68, P3‐X63‐Ag8.653, RAW 264.7, WI‐38, MCF7, BNL CL2, 3T3‐L1, Jurkat (for suitable cell density and amount of medium, refer to Table 2.16.1 for adherent cells or Table 2.16.2 for suspension cells)
  • Appropriate medium for cells
  • 24‐well (or other appropriately sized) culture plates (see Tables 2.16.1 and 2.16.2)
    Table 2.6.1   Materials   Recommended Cell Density for Each Well Plate Size ( protocol 1; for Adherent Cells)   Recommended Cell Density for Each Well Plate Size (; for Adherent Cells)   Recommended Cell Density for Each Well Plate Size ( protocol 1; for Suspension Cells)   Recommended Cell Density for Each Well Plate Size (; for Suspension Cells)

    Plate size Cell density (at inoculation onto the well plate a) Amount of medium
    6‐well plate 0.4−2 × 105 cells/well 2.0 ml/well
    24‐well plate 1−5 × 104 cells/well 0.5 ml/well
    96‐well plate 0.25−1.25 × 104 cells/well 0.125 ml/well
    8‐well chambered coverglass 0.5−2.5 × 104 cells/well 0.2 ml/well
    Cell density/amount of medium
    Inoculation onto well plate
    Plate size Centrifugation (in a tube; step 10b) Medium for resuspension (step 10b) Number of cells Amount of medium
    6‐well plate 0.4−2.0 × 106cells/0.5 ml/tube 2.0 ml 0.4−2.0 × 106cells/well 2.0 ml/well
    24‐well plate 1–5 × 105/0.25 ml/tube 0.5 ml 1.0−5.0 × 105cells/well 0.5 ml/well
    96‐well plate 1−5 × 105cells/0.25 ml/tube 0.5 ml 0.25−1.25 × 105cells/well 0.125 ml/well
    8‐well chambered coverglass 1–5 × 105/0.25 ml/tube 0.5 ml 0.5−2.5 × 105cells/well 0.2 ml/well

     aUsed for transfection under conditions of 1‐day culture and 40% to 60% confluency.
    Table 2.6.2   Materials   Recommended Cell Density for Each Well Plate Size ( protocol 1; for Adherent Cells)   Recommended Cell Density for Each Well Plate Size (; for Adherent Cells)   Recommended Cell Density for Each Well Plate Size ( protocol 1; for Suspension Cells)   Recommended Cell Density for Each Well Plate Size (; for Suspension Cells)

    Plate size Cell density (at inoculation onto the well plate a) Amount of medium
    6‐well plate 0.4−2 × 105 cells/well 2.0 ml/well
    24‐well plate 1−5 × 104 cells/well 0.5 ml/well
    96‐well plate 0.25−1.25 × 104 cells/well 0.125 ml/well
    8‐well chambered coverglass 0.5−2.5 × 104 cells/well 0.2 ml/well
    Cell density/amount of medium
    Inoculation onto well plate
    Plate size Centrifugation (in a tube; step 10b) Medium for resuspension (step 10b) Number of cells Amount of medium
    6‐well plate 0.4−2.0 × 106cells/0.5 ml/tube 2.0 ml 0.4−2.0 × 106cells/well 2.0 ml/well
    24‐well plate 1–5 × 105/0.25 ml/tube 0.5 ml 1.0−5.0 × 105cells/well 0.5 ml/well
    96‐well plate 1−5 × 105cells/0.25 ml/tube 0.5 ml 0.25−1.25 × 105cells/well 0.125 ml/well
    8‐well chambered coverglass 1–5 × 105/0.25 ml/tube 0.5 ml 0.5−2.5 × 105cells/well 0.2 ml/well

NOTE: All materials listed above other than the antibody and cultured cells are contained in the GenomONE‐CAb kit supplied by Ishihara Sangyo Kaisha Ltd. (http://www.iskweb.co.jp/hvj‐e/english‐default.htm). It is possible to prepare these materials oneself according to the recipe and references indicated in Reagents and Solutions, although much skill is required to prepare HVJ envelope or ZZ‐NP of consistent quality.

Alternate Protocol 1: HVJ Envelope‐Mediated Intracellular Delivery of an Affinity‐Tagged Protein (Antibody)

  • Anti‐affinity‐tag antibody (e.g., anti‐Flag, ‐HA, ‐Myc affinity‐tag antibody; also see annotation to step 5), ice cold
  • Affinity‐tagged protein or peptide, ice‐cold
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Figures

Videos

Literature Cited

Literature Cited
   Buchholz, C.J., Spehner, D., Drillien, R., Neubert, W.J., and Homann, H.E. 1993. The conserved N‐terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. J. Virol. 67:5803‐5812.
   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.
   Kondo, Y., Fushikida, K., Fujieda, T., Sakai, K., Miyata, K., Kato, F., and Kato, M. 2008. Efficient delivery of antibody into living cells using a novel HVJ envelope vector system. J. Immunol. Methods 332:10‐17.
   Lukas, J., Pagano, M., Staskova, Z., Draetta, G., and Bartek, J. 1994. Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumour cell lines. Oncogene 9:707‐718.
   Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T.A., and Uhlen, M. 1987. A synthetic IgG‐binding domain based on staphylococcal protein A. Protein Eng. 1:107‐113.
   Sato, M., Iwaya, R., Ogihara, K., Sawahata, R., Kitani, H., Chiba, J., Kurosawa, Y., and Sekikawa, K. 2005. Intrabodies against the EVH1 domain of Wiskott‐Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells. FEBS J. 272:6131‐6144.
   Zelphati, O., Wang, Y., Kitada, S., Reed, J.C., Felgner, P.L., and Corbeil, J. 2001. Intracellular delivery of proteins with a new lipid‐mediated delivery system. J. Biol. Chem. 276:35103‐35110.
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