Our main strategy for reverse genetics is the identification of chemically induced mutations in specific genes. The general method has been called Targeting Induced Local Lesions IN Genomes or TILLING. As a first step we are directly measuring induced mutations in a number of genes by sequencing exon spanning PCR products. We have prepared DNA from ~96 G0 mutagenised tadpoles with corresponding un-mutagenised siblings. Both groups derive from the same clutch of eggs and same sample of sperm, where the sperm sample is split and one?half receives the mutagen treatment. Our analysis for mutations in an exon of BMP4 have yet to reveal any mutations, but the quality of sequence generated thus far has been low and we have adopted a nested primer approach to make the PCR products cleaner. With a robust amplification strategy we should be able to analyse sequence from ~20 genes over the collection of mutagenised tadpole DNA. These data will help us to estimate the number of individual we will need to analyse to identify mutations in any gene.

In the meantime we are freezing testis aliquots from the G0 males generated by the in vitro mutagenesis and IVF described above. Thus far we have stored nearly 50 sets of gametes and somatic tissue for DNA extraction.

We are testing several genotyping methods. Because of the economy of scale offered at the Sanger Institute, we may find that direct sequencing of PCR products is the most sensible approach especially in the short to medium term. The ability to analyse pooled samples using the CelI endonuclease, however, is likely to be the most cost?effective approach in the medium to long term and we are currently optimising the method for use with the ABI 3700 and ABI 3730s most available at the Sanger Institute. To some extent the choice of genotyping method will depend on the efficiency of mutagenesis.

TILLING Scheme
Tilling: Adapted from Colbert et al., (2001). Hight-throughput screening for induced point mutations. Plant Physiol. 126:480-4.