Background:
GenomONE� - CF EX is a cell fusion kit comprised of HVJ Envelope (HVJ-E) and special buffers. It can be used with both adhering cells and suspension cells. Fusion of the same or different cell types can be completed in only 30 minutes.
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GenomONE�
CF EX HVJ Envelope Cell Fusion Kit.
GenomONE� - CF EX is a cell fusion kit comprised of HVJ Envelope (HVJ-E) and special buffers. It can be used with both adhering cells and suspension cells. Fusion of the same or different cell types can be completed in only 30 minutes.
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 |
Principe for cell fusion
triggered by HVJ-E: |
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How HVJ-E Works Membrane Binding: |
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. |
Membrane Distortion: |
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). |
Membrane Fusion:
Cmparison with PEG method in hybridoma preparation
hybridoma growth supplement |
hybridoma positive rate |
antibody-production positive rate |
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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 (1x10^8 cells) not
sensitized with antigen were fused to X63-Ag8.653
myeloma cells (1x10^7 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:
Fusion of adherent cell and suspension cell
GenomONE� - CF
EX Kit Contents |
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Freeze-dried HVJ-E |
0.26 ml |
HVJ-E suspension buffer |
0.5 ml |
20x Cell Fusion Buffer |
10 ml |
_____________________________________________________________________________________________________________________
GenomONE-CF EX HVJ-E 1 Vials Cell fusion Reagents |
ISK-CF-001-EX | 1 SET | �262 |
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