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Hybridomas, Genetic Engineering of 433

To allow fusion the activated B-lymphocytes are mixed which involves the complete synthesis of the purine ring
with a suitable fusion partner (the myeloma) in a medium (called the “de novo” pathway) from smaller metabolic
containing polyethylene glycol (PEG). A proportion of the precursors. Normal cells can utilize either pathway de-
cells will fuse within a minute under these conditions. pending upon the nutrient precursors available in the sur-
rounding media.
X. SELECTABLE GENE MARKERS The myelomas chosen for cell hybridization with lym-
FOR CELL SELECTION phocytes have an HGPRT genetic marker through previ-
−
ous random mutation and selection. However, the hybrido-
The process of cell fusion will result in a heterogeneous mas would be expected to have normal HGPRT activity
+
population of cells that will contain unfused parental cells, (HGPRT ) because they would receive the normal gene
lysed cells as well as the required hybrid cells. At this stage from the parental lymphocyte. Either cell line would be
cell selection is important so that the hybrid cells can be able to grow normally in standard cell culture medium
isolated from the mixture. For hybridomas there are two in which most nutrients are provided. The de novo path-
important stages of cell selection: way for nucleotide synthesis would operate in both cells,
although the salvage pathway would only occur in the
Isolation of hybrid cells from parental cells hybridomas.

Selection of antibody secreting cells within the hybrid The principle of selection is to place these cells into a

cell population. “selective medium” in which the de novo pathway of nu-
cleotide synthesis is inhibited. The key to this is the com-
The basis of cell selection is to distinguish cell types pound, aminopterin which is an analogue of folic acid and
through genetic differences. One required characteristic of a specific inhibitor of dihydrofolate reductase, an essen-
the resulting hybridomas is the ability for effective growth tial enzyme for the formation of tetrahydrofolate (FH 4 )
in culture. So, initial cell selection can involve the incuba- required as a coenzyme of the de novo purine nucleotide
tion of the mixed population of cells in a suitable culture synthesis pathway. Tetrahydrofolate is also required for
environment. This includes the addition of a suitable liq- the formation of thymidine. However, if hypoxanthine and
uid growth medium to the cells and incubation at 37 C for thymidine are provided in the culture media of HGPRT +
◦
a few days. This will allow the growth of all viable cells cells they will be able to grow normally. On the other
−
which will include the hybridomas and myelomas. Growth hand HGPRT cells would have no means of synthesiz-
of these cells will dilute any nonviable and lysed cells from ing purine nucleotides and consequently would be unable
the mixture. Unfused lymphocytes have only a limited ca- to grow.
pacity for growth and will also be eventually eliminated. The selective medium, HAT contains hypoxanthine,
The hybridomas are selected from the myelomas on aminopterin, and thymidine. This medium allows the se-
the basis of a genetic marker that is normally applied to lection and growth of hybridomas which are HGPRT +
the myelomas. The most commonly used genetic marker and have a normal functioning salvage pathway. How-
−
is HGPRT which indicates a cell with a defective en- ever, HAT is unable to support the growth of the HGPRT −
zyme, hypoxanthine guanine phosphoribosyl transferase. myelomas because the de novo pathway is inhibited and
This is a normal metabolic enzyme that is capable of cat- the salvage pathway cannot function because of the de-
alyzing the addition of phosphoribosyl pyrophosphate on fective enzyme.
to hypoxanthine or guanine. This constitutes the salvage In the mixed population of cells resulting from fusion,
+
pathway for converting purines to nucleotides as part of the newly formed hybridomas will be HGPRT by in-
nucleotide synthesis in all normal cells (Fig. 8). However, heritance from normal lymphocytes whereas the unfused
−
there is an alternative pathway of nucleotide formation myelomas carry the mutant HGPRT marker. Therefore
incubation in HAT will allow survival of the HGPRT +
hybridomas but not the HGPRT myelomas. So, after a
−
few days incubation in HAT the culture will contain only
hybridomas.

XI. CLONAL SELECTION OF
Mab-SECRETING HYBRIDOMAS

After genetic selection with HAT the culture contains hy-
bridomas but only some of these will secrete antibodies.
FIGURE 8 De novo and salvage pathways of nucleotide Although the efficiencies of synthesis vary considerably,
synthesis. about 10% of the population of hybridomas formed from