4-5.3.2. Screening by functional complementation
Functional complementation is the process of compensating a missing function in a mutant cell by a particular DNA sequence for restoring the wild-type phenotype. If the mutant cells are non-viable, the cells carrying the clone of interest can be positively selected and isolated. It is a very powerful method of expression cloning and also useful for identification of genes from an organism having same role as that of defective gene in another organism. The selection and identification of positive clones is based on either the gain of function or a visible change in phenotype.
For example, the functional complementation in transgenic mice for the isolation of Shaker-2 gene applied by Probst et al in1988 shown in Figure 4-5.3.2.
Figure 4-5.3.2: Functional complementation in transgenic mice for isolation of Shaker-2 gene.
(Adapted from Primrose SB, Twyman RM. 2006. Principles of gene manipulation and genomics.7th ed. Blackwell Publishing.)
The Shaker-2 mutation is due to the defective gene associated with human deafness disorder. The BAC clone from the wild type mice are prepared and injected into the eggs of Shaker-2 mutants. The resulting mice are then screened for the presence of wild type phenotype. Thus the BAC clone carrying the functional Shaker-2 gene is identified which encodes a cytoskeletal myosin protein. This method can be used for screening human genomic libraries to identify equivalent human gene.