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I, Taniguchi, created a mutant medaka library for TILLING in collaboration with other research institutes, and the system that are necessary to screen and isolate mutants of interest from the library. Several gene disruptants have been reported already (Taniguchi, et al. Genome Biol. 7:R116, 2006）. Because the creation of such mutant library requires a large scale mutagenesis followed by the creation of a huge archive of DNA and sperm, we are the only group in the world who can generate gene knockout medakas.
The schematic for TILLING is shown in Fig. 1.
The TILLING library created by Taniguchi et al consists of about 6,000 medakas mutagenized by an alkylating agent, ENU. If you want to obtain ‘gene A knockout medaka’, the first thing to do is to PCR amplify the specific region of gene A in the search for any mutations contained in that part. We estimate that a mutation can be found in every 60 bases on average throughout the genome if the whole library is screened. This, in other words, means that the average of 10 mutations would be expected to be found for the PCR product of 600 base pair in length. These mutations can be classified into nonsense mutation (an amino acid is converted into a premature termination codon), missense mutation (an amino acid is substituted by others), silent mutation (no change in amino acid), intronic mutation and splice site mutation (the canonical GT-AG rule is disrupted). Among these, the nonsense and splice site mutations, and in some cases the missense mutations, may result in the loss of function of gene A. We recover the fish carrying such mutations by an artificial insemination of cryopreserved sperm.