Dr Siti Suri Lecture 7 Molecular Techniques

PRODUCTION OF MONOCLONAL ANTIBODIES AND CELL FUSION Somatic Cell Fusion Monoclonal antibodies via somatic cell fusion Principles of HAT selection Derivation of HGPRT- and TK- myeloma lines

Uses of somatic cell fusion technology - Monoclonal antibody production Somatic cell genetics - select for genetically somatic cells - map genes to chromososme - Study gene functions-when nucleus are introduced into novel cellular environment. Involve with the Salvage pathways to obtain purine nucleotides and thymidylate TK + cells Convert into 5-BUdr mono phosphate Converted to 5-BUdr triophosphate by other enzymes Inserted by DNA polymerase into DNA Toxic to DNA

SOMATIC CELL FUSION

+

Sendai Virus (enveloped virus that contain fusion proteins, that are made up of hydrophobic aminoacids and therefore can fuse cells Membrane fusion And Nuclear fusion

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

General Procedures-Somatic cell hybridization /fusion Selection of parental cells Select TK- cells ; exposure to BUdR Select HGPRT- cells : exposure to thioguanine (beta-azaguanine) TK+ HGRPT+ BUdR resistance TKHGRP+

TKHGRPT+

TK+ HGRPT-

Normal cells

Beta Azaguanine resistance TK+ HGRP-

TK : Tymidine kinase an enzyme used in the salvage pathway for pyrimidine biosynthesis. The presence of TK is not essential for cells to survive HGPRT : hypoxanthine guanosine phosphoribosyl transferase

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

BUdR resistance

Beta Azaguanine resistance TK- TK+ HGRPT+ HGRPT-

BUdR (thymidine analog) resistance TKHGRPT+

TKHGRPT+

+

PEG (Polyethylene glycol)

Beta Azaguanine resistance TK+ HGRPT-

TK+ HGRPT-

TK+ HGRPT-

TKHGRPT+

Combination of FUSION CELLS and NUCLEUS PEG allow plasma membranes of adjacent cells to adhere to each other and to fuse

After fusion :

TKHGRPT+ = Genetically different cells

TKHGRP+ TKHGRP+

TKHGRPT+

TK+ HGRP-

TKHGRP+

TKTK+ HGRPTHGRPT+

PARENTAL HOMOKARYON (no fusion) ` (two similar parents)

TKTK+ HGRPTHGRPT+

TK+ HGRP-

FUSION OF CELLS

TKTKHGRPT+ HGPRT+

HETEROKARYON (two dissimilar parents)

TK+ TK+ HGRPTHGRPT-

TK+ HGRPT-

HOMOKARYON PARENTAL

SELECTION IN HAT MEDIUM HYBRID CLONE SURVIVING CELLS

TK+ HGRP-

Principle of HAT system HAT medium for selection of hybrids Consist of • Hypoxanthine • Aminopterin and • Thymidine Only hybrid cells that survive in this medium are hybrid cells with TK+ (TK enzyme) and HGPRT+ (HGP enzyme) Hybrid cells: Cells formed by the fusion of two different cells (heterokaryons)

MONOCLONAL ANTIBODIES PRODUCTION USING SOMATIC CELL FUSION Hybridoma technology by kohler and milstein 1975 Making monoclonal antibodies Prepare highly purified and cintrated antigen 1 Immunization of mice with antigen by the intraperitoneal route 2 When serum shows sufficient antibodies, the mouse is killed and the spleen harvested 3 Spleen is cut up and made into a cell suspension 4 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 or are kept for use in liquid nitrogen 11 Intraperitoneal injection of the clones induces antibody rich ascitic fluid in mice

Hybridoma cells grown in 96 well

Hybridoma cells producing antibody

Ascitic fluid containing high antibody After inoculation with hybridoma cells

+

TKHGRPMYELOMA CELLS

PEG (Polyethylene glycol)

Non secreting Transformed and immortal cells

TK+ HGRP+ SPLEEN CELLS Short survival Survive a few days only TK- and HGPRT + Produced antibodies

TK+ and HGPRT – (necessary for DNA synthesis Cannot survive HAT medium

TKHGRPT-

TKTKHGRPTHGPRT-

TKTK+ HGRPTHGRPT+

TK+ TK+ HGRPT+ HGRPT+

HYBRIDOMA CELLS CLONE IN HAT MEDIUM

TK+ HGRPT+

TKTK+ HGRPTHGRPT+

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

Selection for the hybridomas. The myeloma cells are HGPRT- and the B cells are HGPRT+. HGPRT is hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in 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 HAT medium since they are HGPRT-. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous.

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 formed and taken up by the cells via phagocytosis (2) Cationic liposome –mediated transfection Using lipofectin -polycationic lipid reagent -ionic interaction of DNA and liposomes to form a complex to deliver DNA into Cultured 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

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

Briefly exposing high cell concentration to a high voltage electricfield in the presence of DNA to be transfected. Small holes are generated transiently and the DNA is allowed to enter and in some of the cells, the DNA becomes incorporated into the genome.

DNA Transfer