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Superior to PEG for hybridoma preparation and other cell fusion procedures

GenomONE™ - CF EX Sendai virus (HVJ) Envelope Cell Fusion Kit

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Background

GenomONE-CF EX is a cell fusion kit composed of HVJ Envelope (HVJ-E) and special buffers.
It can be used with both adhering cells and floating cells. Fusion of cells of the same or different types is possible with this kit in only 30 minutes.

Experimental Protocol  movie Watch the video


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Principle

What is HVJ Envelope (HVJ-E)?

HVJ Envelope is a purified product prepared through complete inactivation of Sendai virus (HVJ*). It is a vesicle in which only the cell membrane-fusing capability of the envelope protein of Sendai virus is retained.


It is known that the HN protein in the tunica externa of the Sendai virus recognizes the receptor (possessing sialic acid at the terminal of sugar chain) on the cell membrane and adsorbs it, leading to the induction of membrane fusion mediated by F protein (another component of the envelope). The genomic RNA of the Sendai virus contained in HVJ-E has been inactivated completely and has neither infective nor proliferative potential in humans or experimental animals. HVJ-E can be used safely at ordinary laboratories, without requiring any special operations or facilities
*HVJ:Hemagglutinating Virus of Japan

Animation of cell fusion

Principle for cell fusion triggered by Sendai virus envelope (HVJ-E)


If HVJ-E is added in amounts of more than several hundred HVJ-E per cell at low temperatures (0-8°C), HVJ-E is immediately adsorbed on the cell surface mediated by the receptor (acetyl type sialic acid recognized by HN protein) (Membrane Binding), and cells undergo agglutination cross-linked by HVJ-E particles (Membrane Distortion). At this stage, the hydrophobic domain at the N-terminal of cleaved F protein (F1) penetrates into the double lipid layer of the cell membrane, causing distortion of the membrane severe enough to allow an inflow of ions.

If this cell/HVJ-E complex is heated at 37°C, the distortion of the cell membrane is further expanded, accompanied by temporary alteration of the cell membrane lining structure.  This change is transient and the membrane soon returns to its normal structure.   However, if a strong hydrophobic connective force is applied at this stage, fusion between cell membranes takes place (Membrane Fusion).

Features and Advantages

Cmparison with PEG method in hybridoma preparation

  hybridoma
growth supplement
hybridoma
positive rate
antibody-production
positive rate
GenomONE - CF EX - 38/96 (40%) 9/96 (9%)
+ 96/96 (100%) 96/96 (100%)
PEG1500 - 3/96 (3%) 1/96 (1%)
+ 36/96 (38%) 9/96 (9%)

GenomONE - CF EX produces more antibody secreting hybridomas than PEG. (following cell fusion, cells were grown in media containing hybridoma growth supplement as indicated).

「Normal BALB/c mouse splenocytes (1x10ˆ8 cells) not sensitized with antigen were fused to X63-Ag8.653 myeloma cells (1x10ˆ7 cells) using GenomONE(tm)-CF EX  or PEG1500. The fused cells obtained with each agent were inoculated onto five 96-well plates (Day 0). Beginning the following day, half of the culture medium (10%FBS/RPMI1640) was replaced with HAT medium at five points of time (Days 1, 2, 3, 5, and 8), and the growth of colonies in each well was assessed on Days 10 -11 to determine the hybridoma-positive rate
(an indicator of efficiency of fusion). On Day 12, mouse antibody level (IgG + IgA + IgM) in the supernatant was measured by ELISA, to calculate the antibody production-positive rate. The effect of adding a commercially available hybridoma supplement to the medium after fusion was also assessed (supplement was also added to the HAT medium).

Footnote:
The results shown above are an example, and experimental conditions need to be optimized depending on the type of antigen, myeloma cell, supplement etc. used.

Application


GenomONE-CF Application

GenomONE-CF EX fusion protocol of donor cells to mouse enucleated oocytes
GenomONE-CF References
GenomONE (HVJ-Envelope)
References (Cell Fusion) 
  1. Hybridoma preparation
    Cell fusion involving PEG and electrofusion Preparation of monoclonal antibodies Fusion of antigen-sensitized B cells and myeloma cells

    Example (Published research article)
    Generation and Characterization of Novel Monoclonal Antibodies Against Murine and Human TARSH Proteins.
    N. Uekawa et al . Hybridoma (Larchmt), 26, 381-385(2007).PMID:18158782

  2. Fusion of suspension cells
    Example (Published research article)
    Enhanced Tumor-Specific Long-Term Immunity of Hemaggluttinating Virus of Japan-Mediated Dendritic Cell-Tumor Fused Cell Vaccination by Coadministration with CpG Oligodeoxynucleotides
    K. Hiraoka et al. J. Immunol.,173, 4297-4307(2004).PMID:15383558

  3. Fusion of adherent cell and suspension cell

  4. Application in premature chromosome condensation (PCC)
    Detection of chromosomal damage and repair by fusion of different types of cells in different cell phases

  5. Research on regenerative medicine and cytotherapy
    Fusion of somatic cells and stem cells, etc.

  6. Research on embryonic development/differentiation
    Nuclear transfer (nuclear replacement)

  7. GenomONE - NEO EX HVJ Envelope Vector Kit
    Unique, efficient transfection of siRNA, DNA, oligonucleotides, proteins.
    Low toxicity with primary cell lines.

Protocol

GenomONE-CF protocol

GenomONE - CF EX Instruction Manual-PDF PDF

Frequently Asked Questions

Go to FAQ Page: Frequently asked Questions

Kit component

GenomONE - CF EX Kit Contents
(for 10 hybridoma fusion experiments)

Freeze-dried Sendai virus envelope (HVJ-E) 0.26 mL
equivalent
Sendai virus envelope (HVJ-E) suspension buffer 0.5 mL
20x Cell Fusion Buffer 10 mL

Product List

Product Name Cat# Quantity Price

GenomONE-CF EX SeV-E (HVJ-E) 1 vial Cell Fusion Reagents

ISK-CF-001-EX 1SET

¥ 25,000
$ 334
€ 250

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.