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GenomONE™ - CF EX

Sendai Virus (HVJ) Envelope Cell Fusion Kit

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.

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 (component of 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 animals. HVJ-E can be used safely in standard laboratories, without requiring special procedures or facilities.
*HVJ:Hemagglutinating Virus of Japan.

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
  Hybridoma growth supplement Hybridoma positive rate Antibody-production positive rate
GenomONE™ - CF EX - 38/96 (40%) 9/96 (9%)
GenomONE™ - CF EX + 96/96 (100%) 96/96 (100%)
PEG1500 - 3/96 (3%) 1/96 (1%)
PEG1500 + 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 (1x10^8 cells) not sensitized with antigen were fused to X63-Ag8.653 myeloma cells (1x10^7 cells) using GenomONE™ - 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).

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