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#cell fusion

17 public questions tagged with this topic.

Plant tissue culture 2

Which of the following is NOT a fusogen?

Colchicine is not considered fusogen, unlike true fusogenic agents that promote membrane merging of protoplasts. Legitimate fusogens include polyethylene glycol PEG high molecular weight polymer that dehydrates membrane interface creating calcium bridges neutralizing negative charge inducing lipid mixing upon dilution, high concentration calcium ions at alkaline pH around 10.5 similar mechanism reducing electrostatic repulsion, and inactivated Sendai virus bearing fusogenic HN and F glycoproteins that merge animal and plant membranes albeit rarely used now due to biohazard handling. Electrical

Ref: Anne & Harada 1991 fusogens list; Kao 1977 colchicine not fusogen.

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Plant tissue culture 2

Somatic hybridization can transfer:

Somatic hybridization can transfer extensive genetic material ranging from blocks of linked genes to entire chromosomes or whole genomes, contrasting with single gene transformation using plasmid vectors. When two protoplasts fuse, initial heterokaryon contains cytoplasm of both parents and nuclei that may fuse producing allotetraploid somatic hybrid carrying full complement of chromosomes from both species, allowing introgression of polygenic quantitative trait loci QTL controlled by multiple genes interacting for disease resistance, abiotic stress tolerance, quality attributes that cannot be

Ref: Helgeson 1979 potato somatic hybrid; Bhat & Bhat 2011 gene blocks transfer.

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Plant tissue culture 2

Electrofusion induces fusion by:

Electrofusion induces protoplast fusion by applying controlled electrical pulses that create reversible membrane breakdown at points of contact allowing lipid bilayers to merge and cytoplasms to coalesce, offering higher fusion frequency viability and selectivity than chemical PEG method. Protocol consists of two electrical phases: first low strength high frequency alternating current 50 to 200 kilohertz field causes dielectrophoresis polarizing protoplasts forming induced dipoles aligning them in pearl chains bringing membranes into intimate contact at poles. Second short high intensity direc

Ref: Zimmermann & Vienken 1982 electrofusion; Tempelaar et al., Plant Sci electroporation.

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Immunological Applications

Myeloma cells used in hybridoma technology are selected to be:

Establishing stringent selection requires myeloma background auxotrophic for purine salvage, achieved by exposure to 8-azaguanine, purine analogue structurally similar to guanine. Salvage competent cells convert 8-azaguanine via HGPRT to 8-azaguanosine monophosphate incorporated into mRNA and rRNA causing miscoding, translational errors, and lethal proteotoxicity triggering unfolded protein response. Spontaneous HPRT1 mutants survive because they cannot metabolize analogue, accumulating no toxic nucleotides. Surviving subclones harbor frameshift, nonsense, or missense mutations in coding exons

Ref: Szybalski & Szybalska 1962 HGPRT negative mutants 8-azaguanine; ATCC SP2/0 Ag14 datasheet selection mechanism.

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Immunological Applications

PEG (polyethylene glycol) is used in hybridoma technology for:

Efficient cell fusion must overcome hydration repulsion keeping lipid bilayers approximately two nanometers apart due to ordered water molecules bound to phosphatidylcholine headgroups. Polyethylene glycol with molecular weight 1450 to 4000 daltons is extremely hygroscopic linear polymer containing repeating ether oxygens that strongly bind water through hydrogen bonding, lowering water activity dramatically and creating osmotic stress that forces membranes into close proximity below one nanometer. At concentrated 40 to 50 percent weight per volume, PEG disrupts lipid packing, induces inverted

Ref: Lentz J Mol Biol PEG fusion membrane dehydration; Alberts MBoC membrane fusion hemifusion stalk; Nature Protocols PEG hybridoma.

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Immunological Applications

Hybridoma technology involves fusion of:

Hybridoma formation relies on merging two complementary cellular programs within one cytoplasm to create self-renewing antibody factory. Splenic B lymphocytes from immunized donor possess productively rearranged heavy chain locus on chromosome 14 with VDJ recombination mediated by RAG1/2 and light chain kappa or lambda locus with VJ recombination, encoding high-affinity combining site selected through germinal center affinity maturation involving activation-induced deaminase, but they senesce within days after extraction due to lack of telomerase, dependence on BAFF survival signal, and activa

Ref: Janeway Immunobiology 9th ed monoclonal generation; J Genet Eng Biotechnol 2020 PMC7414427 hybridoma fusion B myeloma.

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Immunological Applications

PEG (polyethylene glycol) is used in hybridoma technology for:

Polyethylene glycol polymer repeating ethylene oxide units average MW 1500 Da widely employed fusogen hybridoma generation due unique physicochemical properties affecting membrane biophysics. Mechanism involves strong hygroscopic nature binding water around phospholipid headgroups phosphatidylcholine sphingomyelin reducing hydration repulsion normally maintaining 2-3 nm separation bilayers enabling close apposition

Ref: Kohler Milstein PEG Fusion Method 1976 Detailed; Alberts Membrane Fusion PEG Mechanism Biophysical; Janeway Hybridoma PEG Fusion Laboratory Protocol Chapter 5.

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Immunological Applications

Hybridoma technology involves fusion of:

Somatic cell fusion underlying hybridoma merges two differentiated cell types each contributing specialized traits necessary monoclonal antibody manufacturing. Antigen-experienced B lymphocyte harvested spleen four days after final booster carries functionally rearranged Ig heavy VDJ and light VJ genes formed RAG1/RAG2 recombination junctional diversity somatic hypermutation conferring high affinity specificity target epitope active transcription intronic enhancer Emu 3 prime regulatory region capacity synthesizing Ig up to 2000 molecules per second but limited lifespan due lack telomerase dep

Ref: Janeway Hybridoma Fusion B cell Myeloma Mechanism 9th ed; Alberts Cell Fusion Mechanism Chapter 19; NCBI Kohler Milstein Hybridoma Methodology Original.

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