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 7-2, but is specific for the CHO Cell BrdU-VL system. In this technique, 

 CHO-Kl cells are cultured in two types of media. One medium (F12D) 

 contains the minimal nutritional requirements for the growth of single cells 

 into macroscopic colonies with high efficiency. A second, enriched medium 

 (F12) is constructed from the minimal by addition of nine nutrilites (alanine, 

 glycine, aspartic acid, glutamic acid, lipoic acid, vitamin Bj2, inositol, 



A B C 



chemical 

 treatment 



mutation 

 induction 



mutant cell 

 selection 



■> 



5-bromodeoxyuridine 



colony of 



mutant 



cells 



exposure 



to 



white 



light 



mutant cell growth vj/ 



< © 



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)). 



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