Cell fusion involving PEG and electrofusion
Preparation of monoclonal antibodies
Fusion of antigen-sensitized B cells and myeloma cells
|Immediately after mixing||After one day of culturing||After 2 days of culturing|
|GenomONE - CF EX|
Rat MSC cells (rat bone marrow-derived mesenchymal stem cells) labeled with red fluorescence*1 were combined with rat primary cardiac myocytes labeled with green fluorescence*2 (each 2 x 105 cells) in 50 μL of a buffer for cell fusion. The mixture was incubated on ice for 5 minutes and then at 37°C for 15 minutes. As a result, fused cells (yellow) were formed (GenomONE - CF EXsuspension method). Fused cells adhering to the plate were also observed after 1-2 days of culturing. In the PEG-treated group, high cytotoxicity appeared immediately after cell fusion, reducing the number of fused cells obtained
(*1) Labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma)
(*2) Labeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma)
|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).
The footpad of mice was immunized with KLH three times (on Day 1, 4 and 7). Three days after the last immunization (Day 10), lymphocytes were collected from the lymph nodes of these mice. These lymphocytes were combined with myeloma cells and the mixture treated with HVJ-E or PEG (molecular weight: 3,000-3,700) to induce cell fusion. Twenty-eight days later, proliferation of hybridomas was checked and wells showing formation of KLH-specific antibody were counted (ELISA).
Data supplied by Dr. Masahiro Tomita, Laboratory of Molecular Bioengineering, Department of Chemistry for Materials, Faculty of Engineering, Mie University, Japan.
Depending on the type of antigens or myeloma cells used, the conditions for fusion shown in the above examples may require some optimization.
Cells labeled with red fluorescence*1 were combined with cells labeled with green fluorescence*2 (each 1-2 x105 cells) in a fusion buffer (50μL). HVJ-E was added to the mixture, followed by incubation on ice for 5 minutes and further incubation at 37°C for 15 minutes. Fused cells (yellow) were obtained.
Cells labeled with red fluorescence*1 adhering to a 12-well plate were combined with 1 × 105 suspension cells labeled with green fluorescence*2 and pre-treated with HVJ-E (suspended in fusion buffer). Each plate was centrifuged at 1000 rpm at room temperature for 5 minutes. This was followed by incubation at 37 °C for 15 minutes. The medium was then replaced with a medium for cell proliferation, and the cells were incubated further. Fused cells (yellow) were observed 3 hours later.
Fusion of adhering neonatal rat primary cardiac myocytes and floating P19 cells (GFP-expressed cells)
2.5 x 105 P19 cells pretreated with HVJ-E (emitting green fluorescence due to GFP gene transfection) were added to rat primary cardiac myocytes adhering to a 12-well plate. Each plate was centrifuged at 1000 rpm at 20°C for 3 minutes. The medium was then replaced with a medium for cell proliferation, and the cells incubated further. Twenty-four hours later, cells showing strong GFP expression due to fusion with P19 cells were found among the pulsatile myocytes in the HVJ-E treated wells.
Data supplied by Dr. Hironori Nakagami,
Division of Gene Therapy Science, Graduate School of Medicine,
Osaka University, Japan.
*1: For fluorescent labeling, PHK26 Red Fluorescent Cell Linker Kit (Sigma) was used.
*2: For fluorescent labeling, PKH67 Green Fluorescent Cell Linker Kit (Sigma) was used.
Detection of chromosomal damage and repair by fusion of different types of cells in different cell phases
If cells in M phase (during mitosis) are fused with cells in G0/G1 phase (inter-phase), the fused cells show both M phase chromosomes and G0/G1 phase chromosomes. Usually, G0/G1 phase chromosomes are not visible because they have not undergone condensation. However, MPF (M phase promoting factor) in the cytoplasm in M phase can induce premature chromosome condensation (PCC), resulting in visualization of G0/G1 phase chromosomes. By utilizing this phenomenon, it is possible to check G0/G1 phase chromosomes for damage and repair. GenomONE - CF EX was used for preparing the fused cells employed in PCC.
Data supplied by Dr. Maki Okada and Dr. Ryuichi Okayasu,
International Space Radiation Laboratory, National Institute of Radiological Sciences, Japan.
References: Okada. M., et al., Mutation Research, 562 (1-2), 11-17 (2004).
Okayasu. R., et al., Radiation Research, 165 (1), 59-67 (2006).
Fusion of somatic cells and stem cells, etc.
Nuclear transfer (nuclear replacement)
Unique, efficient transfection of siRNA, DNA, oligonucleotides, proteins. Low toxicity with primary cell lines.
GenomONE-CF EX SeV-E (HVJ-E) 1 vial Cell Fusion Reagents