GENERALIZED AND SIMPLIFIED PROTOCOL FOR POINT MUTATION IN CELLULAR SYSTEMS 
CELL INOCULATION 
AND 
EXPOSURE TO 
TEST AGENT 
MUTANT 
EXPRESSION 
MUTANT CELL 
SELECTION 
MUTANT CELL 
ANALYSIS 
ATTACHMENT 
WASH 
Figure 7-2. A generalized and simplified protocol for the 
induction, isolation and characterization of mutations 
in cellular systems. 
Figure 7-3 represents a protocol containing the same basic features as that in 
Figure 7-1, but is specific for the CHO Cell/BrdU-VL system. In this technique, 
CHO-K1 cells are cultured in two types of media. One medium (FI2D) 
contains the minimal nutritional requirements for the growth of single cells 
into macroscopic colonies with high efficiency. A second, enriched medium 
(FI2) is constructed from the minimal by addition of nine nutrients (alanine, 
glycine, aspartic acid, glutamic acid, lipoic acid, vitamin B^, inositol, 
A B C 
chemical 
treatment 
mutation 
induction 
mutant cell 
selection 
5 -bromodeoxyuridine 
exposure 
to 
white 
light 
colony of 
mutant = 
cells 
mutant cell growth 
<- 
E 
D 
Figure 7-3. Schematic representation of the Chinese hamster 
ovarian cell system (BrdU-visible light selection procedure) for 
the detection of small-scale mutations. 
Note: (A) Population of wild-type cells. (B) Nutritional mutant (auxotroph) 
following induction and expression. (C) Wild-type cells with BrdU-containing 
DNA. (D) Surviving mutant cell following selective elimination of wild-types 
via the combined action of BrdU and white light. (E) Colony of mutant cells 
which grew in enriched medium. (After Kao and Puck (28)). 
83 
