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Somatic Cell Fusion

Monoclonal antibodies via somatic cell fusion

Principles of HAT selection

Derivation of HGPRT- and TK- myeloma lines

PRODUCTION OF MONOCLONAL ANTIBODIES AND CELL FUSION

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SOMATIC CELL FUSION

+ Sendai Virus(enveloped virusthat contain fusion proteins,

that are made up ofhydrophobic aminoacids

and therefore can fuse cells

Membrane fusionAndNuclear fusion

Nuclear fusion: produce viable cells that contains chromosome from both parents

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General Procedures-Somatic cell hybridization /fusion

Selection of parental cells

Select TK- cells ; exposure to BUdRSelect HGPRT- cells : exposure to thioguanine (beta-azaguanine)

TK-HGRPT+ Normal cells

TK+HGRP-

TK-HGRP+

BUdRresistance

Beta Azaguanineresistance

TK : Tymidine kinase an enzyme used in the salvage pathway forpyrimidine biosynthesis. The presence of TK is not essential for cells to survive

HGPRT : hypoxanthine guanosine phosphoribosyl transferase

TK+

HGRPT-

TK+

HGRPT+

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Somatic cell hybridization /fusion

Selection of parental cells

Select TK- cells ; exposure to BUdR (5 bromodeoxyuridine )Select HGPRT- cells : exposure to thioguanine

TK+

HGRPT+

Normal cells

TK+HGRPT-

TK-HGRPT+

BUdRresistance

Beta Azaguanineresistance

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BUdR (thymidine analog)resistance

Beta Azaguanineresistance+

Combination of FUSION CELLS and NUCLEUS

TK+HGRPT-

TK-

HGRPT+

PEG

(Polyethylene glycol)

TK-HGRPT+

TK-HGRPT+

TK+HGRPT-

TK+HGRPT-

PEG allow plasma membranes of adjacent cells to adhere toeach other and to fuse

TK-

HGRPT+ Genetically different cellsAfter fusion : =

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TK+TK+HGRPT-HGRPT-

TK-HGRPT+

TK+HGRPT-

TK-TK-HGRPT+HGPRT+

TK-TK+HGRPT-HGRPT+

TK-HGRP+

TK-HGRP+

TK-HGRP+

TK+HGRP-

TK+HGRP-

TK+HGRP-FUSION OF CELLS

PARENTAL HOMOKARYON HETEROKARYON HOMOKARYON PARENTAL(no fusion) ` (two similar (two dissimilar

parents) parents)

SELECTION IN HAT MEDIUM

HYBRID CLONE

SURVIVING CELLS

TK-TK+HGRPT-

HGRPT+

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Principle of HAT system

HAT medium for selection of hybridsConsist of Hypoxanthine Aminopterin and Thymidine

Only hybrid cells that survive in this medium are hybrid cellswith TK+ (TK enzyme)and HGPRT+ (HGP enzyme)

Hybrid cells:Cells formed by the fusion of two different cells (heterokaryons)

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Making monoclonal antibodies

Prepare highly purified and cintrated antigen

1 Immunization of mice with antigen by the intraperitoneal route2 When serum shows sufficient antibodies, the mouse is

killed and the spleen harvested3 Spleen is cut up and made into a cell suspension4 Mouse myeloma cells from a culture are added to the 5 Spleen cells and the cell mixture is fused by slowly adding ...6 Polyethylene glycol (P.E.G.)7 The fused cells are grown in microplates or in ...8 Soft agar. Cell colonies which produce the desired antibodies are ...9 Selected and passaged a few times to get stable clones which are .

10 Kept in tissue culture for production of monoclonal antibodies orare kept for use in liquid nitrogen11 Intraperitoneal injection of the clones induces antibody rich

ascitic fluid in mice

MONOCLONAL ANTIBODIES PRODUCTIONUSING SOMATIC CELL FUSION

Hybridoma technology by kohler and milstein 1975

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Hybridoma cells grown in 96 well

Hybridoma cells producing antibody

Ascitic fluid containing high antibodyAfter inoculation with hybridoma cells

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TK+HGRP+

TK-HGRP-

MYELOMA CELLS SPLEEN CELLS

Non secretingTransformed and immortal cells

TK+ and HGPRT

(necessary for DNA synthesis

Cannot survive HAT medium

Short survivalSurvive a few days onlyTK- and HGPRT +Produced antibodies

+PEG(Polyethylene glycol)

TK+TK+HGRPT+HGRPT+

TK-HGRPT-

TK+HGRPT+

TK-TK-HGRPT-HGPRT-

TK-

TK+HGRPT-HGRPT+

HYBRIDOMA CELLS

CLONE IN HAT MEDIUM

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TK-

TK+

HGRPT-

HGRPT+

HYBRIDOMA CELLS

Genetic information from both parents

Survive HAT selection medium

Cultured indefinitely

Produce antibody (monoclonal) in the supernatant

-bulk-homogenous- antibody against one epitope only

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Selection for the hybridomas.

The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRTis

hypoxanthine-guanine phosphoribosyl transferase, an enzyme involvedin the synthesis of nucleotides from hypoxanthine, an amino acid.

The culture is grown in HAT(hypoxanthine-aminopterin-thymine)medium, which can sustain only HGPRT+ cells. The myeloma cells that

fuse with another myeloma cell or do not fuse at all die in the HATmedium since they are HGPRT-.

The B cells that fuse with another B cell or do not fuse at all diebecause they do not have the capacity to divide indefinitely. Only

hybridomas between B cells and myeloma cells survive, being bothHGPRT+ and cancerous.

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TRANSFERING GENES INTO MAMMALIAN CELLS

(1)Coprecipitation with CaPO4 Calcium phosphate

Exogenous DNA is mixed with CaCl and and is added to a solution

containing phosphate ions A Ca-PO4-DNA co-precipitate is formedand taken up by the cells via phagocytosis

(2) Cationic liposome mediated transfectionUsing lipofectin -polycationic lipid reagent

-ionic interaction of DNA and liposomes to form a complex to deliver DNA intoCultured cells

Higher efficiency compared to the other methods-the ability to transfect a wide variety of eucaryotic cell lines

-low cell toxicity-Cells transfected with RNA, synthetic oligonucleotide, protein aad vjruses-Delivery of functional genes or viral genomes

Disadvantage:

High cost which practically precludes large scale use

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(3) Electroporation(The equipment : the electroporator is available commercially)

Briefly exposing high cell concentration to a high voltage electric-field in the presence of DNA to be transfected.

Small holes are generated transiently and the DNA is allowed toenter and in some of the cells, the DNA becomes incorporated intothe genome.

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DNA Transfer

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