Fusion of cells by peg and inactivated virus
- 1. Done by : Lamya Alsadhan Submitted to : Dr. Elsayed Ahmed Elsayed
- 2. Objectives: •What is cell fusion? •Types of Cell Fusion •The Advantages and Disadvantages of Cell Fusion. •Methods of Cell Fusion: • PEG. • E-Fusion. • SENDAI virus.
- 3. What is Cell fusion? Cell fusion is an important cellular process in which several uninuclear cells (cells with a single nucleus) combine to form a multinuclear cell, known as a SYNCYTIUM.
- 4. Brief History About Cell Fusion • 1847 : Theodore Schwann first provided hint of cell fusion. • 1960 : cell biologists started deliberately fusion of cells using the Sendai virus. • late 1960s : biologists successfully fused cells of different types and from different species. • 2002 : the possibility of cell fusion between cells of different types may have a real function in mammals.
- 6. Types of Cell Fusion: • Homotypic cell fusion: occurs between cells of the same type. • An example of this would be osteoclasts being fusing together with their respective type of cells. Potential mechanism of fusion of preosteoclasts and of macrophages. Agnès Vignery J Exp Med 2005;202:337-340 © 2005 Rockefeller University Press
- 7. Types of Cell Fusion: •Heterotypic cell fusion occurs between cells of different types, making it the exact opposite of homotypic cell fusion.
- 8. TheAdvantages of Cell Fusion • Help in mapping of chromosomes. • Help in generation/production of monoclonal antibodies. • To create new species. • Cut the species barrier genetically that cant be done through the fusion of gametes. The Disadvantages of Cell Fusion • Low genetic stability and high risk of contamination. • The quality and quantity of antibodies produced are low. • Expensive, as it uses materials and equipment's for mammalian cell culture. • Genetic engineering can’t be done.
- 9. Methods of Cell Fusion: • There are three methods that cell biologists use to fuse cells. These three ways include Polyethylene glycol (peg) cell fusion, Electrical cell fusion and Sendai virus induced cell fusion.
- 10. What is POLYETHYLENE GLYCOL (PEG)? • Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. • PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H−(O−CH2−CH2)n−OH.
- 11. WhatActually PEG Do to Fuse the Cells: (1) Altering the structure and dielectric properties of water. (2) Altering the molecular order of the bilayer at the point of contact due to aggregation and dehydration. (3) Induction of non-bilayer structures (4) Acting as a detergent to disrupt bilayer structure (5) Binding to and cross-linking vesicles. (6) Inducing phase separation that destabilizes the bilayer .
- 12. Biological Applications of PEG: • Commonly used as a crowding agent in in vitro assays to mimic highly crowded cellular conditions. • PEG is commonly used as a precipitant for plasmid DNA isolation and protein crystallization. • PEG is used to fuse two different types of cells, most often B-cells and myelomas in order to create HYBRIDOMAS. • In microbiology, PEG precipitation is used to concentrate viruses. • PEG is also used to induce complete fusion (mixing of both inner and outer leaflets) in liposomes reconstituted in vitro. • Gene therapy vectors (such as viruses) can be PEG-coated to shield them from inactivation by the immune system and to de-target them from organs where they may build up and have a toxic effect. • In blood banking, PEG is used as a potentiator to enhance detection of antigens and antibodies.
- 13. Consequences of Cell Fusion with POLYETHYLENE GLYCOL (PEG) • Polyethylene glycol cell fusion is the simplest, but most toxic, way to fuse the cells. • PEG acts as a dehydrating agent and fuses not only plasma membranes but also intracellular membranes. • PEG can cause uncontrollable fusion leading to the appearance of giant polykaryons. • poorly reproducible.
- 14. Electrofusion (E-fusion) • E-fusion uses electricity instead of chemicals to align and fuse cells.
- 15. Applications : •Nuclear transfer •Embryo manipulation •Hybridoma production •Plant protoplast fusion
- 16. How E-fusion compared to PEG: • E-fusion produces 10 fold higher fusion efficiencies compared to PEG. • E-fusion is not cytotoxic to cells. PEG causes H202 buildup which is highly toxic to cells. • E-fusion is optimized and reproducible, PEG is difficult to work with and results fluctuate from experiment to experiment.
- 17. Sendai virus induced cell fusion : We Must First Understand HowVIRUS Gain Entry into Cells
- 18. Sendai virus (SeV): • also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan (HVJ). • Sendai virus (SeV) was first isolated in Sendai, Japan. • SeV is single-stranded RNA virus • SeV is a member of genus Respirovirus, members of which primarily infect mammals. • SeV used in the fusion of eukaryotic cells, especially to produce HYBRIDOMA
- 19. SENDAIVIRUS Components Helps in Cell Fusion • Six genes coding for viral proteins are situated sequentially on the genome of the wild-type SeV in the following order: • Nucelocapsid protein (NP) • Phosphoprotein (P) • Matrix protein (M) • Fusion protein (F) • Hemagglutinin-Neuraminidase (HN) • Large protein (L)
- 20. How SENDAIVIRUS Fuses with Cell Membrane • One way of SeV infection of cells by attaching itself to the sialic acid receptor or sialic acid residues on gangliosides (glycolipids) on cell receptor and HN protein on the virus membrane. • Another way is by the activation of F protein by a protease is required for the virus-cell fusion process to take place. • Upon binding of the virion to a cell surface receptor, the F1/F2 (fusion) protein undergoes a conformational change at neutral pH that allows insertion of the fusion peptide into the plasma membrane of the cell. This brings the virus envelope close to the cell membrane and leads to fusion of the two membranes, releasing the nucleocapsid in the cytoplasm. • When the HN and F protein are manufactured by the cell and expressed on the surface, the same process may occur between adjacent cells, causing extensive membrane fusion and resulting in the formation of a syncytium
- 21. Why Inactivation of SeVVIRUS is Necessary • UV rays can be used, however, to inactivate viruses since virus particles are small and the UV rays can reach the genetic material, inducing the dimerisation of nucleic acids. Once the DNA dimerised, the virus particules cannot replicate their genetic material which prevent them from spreading.