Xenopus tropicalis Mutation Resource

Tilling Pipeline

The genes to be targeted will be requested by the community through a web page similar to the Zf Mutant Resource Project.

The structure of the gene will be obtained through ensemble and the evidences supplied by the requestor.

Then a series of small amplicons are selected according to their pupative mutation rates. From our Zf Mutant Resource project we have obtained a spectrum of nucleotide changes similar to what has been published before. We use this nucleotide mutation spectrum to evaluate the likelyhood of obtaining a nonsense mutation throughout the coding sequence. In addition we evaluate the probability of producing a Splice site mutant. These probabilities are used to select the best amplicons to sequence. All these analysis will be performed by a modified version of the Limstill software, a web based application developed at the Edwin Cuppen Lab in the Hubrecht Laboratory, Holland. This software also designs the primers to perform a nested PCR. This design will rely on the primer 3 tool developed by Steve Rozen and Helen Skaletsky at the Whitehead_Institute and the Howard Hughes Medical Institute.

The nested PCRs for a partilular amplicon will be performed with the genomic DNA from the mutagenized library.

Figure 1.

Figure 1.

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The sequences will be analyzed by the Mutation Finder software developed by Derek Stemple. This software first filters the sequences based on their quality. Then the program compares all against the reference Genbank-formatted file containing exon information. The program is able to detect changes in their heterozygous state by analyzing size and shape of each peak. It also evaluates the AA change and displays the results graphicaly for manual curation. Individual traces will be categorized as potential heterozygous mutations if they displayed both a reduced height of a wild-type peak and no background noise throughout the trace.

The putative candidates will be confirmed by re-sequence a new nested PCR from the library.

If the mutation is confirmed, the carrier will be recovered by genotyping each individual of the tank if it is alive or directly from the frozen sperm.

An F3 generation will be produced for posterior characterization (see Phenotyping pipeline).

Figure 2.

Figure 2.

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As a pilot experiment we screened 5 amplicons in a library of 1395 samples of 5-day-old F2 tadpoles genomic DNA produced from 65 F1 mosaic adults(See table). We confirmed five nonsense mutations in duplicate TILLING experiments, which we selected for further analysis. Four of these mutations were observed in 1/24 siblings screened, and are indicated in Table 2, but the fifth, isolated in separate TILLING experiments, a mutation in nfat (Nuclear factor of activated T cells) (Shaw et al., 1988) was detected in 5 siblings out of 24. The cognate mosaic F1 frozen sperm samples were used to carry out in vitro fertilization. Among the four mutations that were observed in only 1/24 siblings, one mutation was unrecoverable due to poor frozen sperm quality. Three others were not recovered despite screening more than 90 embryos, consistent with a high degree of mosaicism in the F1 male. However, the nfat nonsense mutation initially appeared less subject to mosacism, and indeed was identified in 3 out of 16 tadpoles recovered from frozen sperm, a similar frequency to that observed in the screen. As this mutation deletes the carboxy terminal two thirds of the protein, it is likely to result in a loss of function phenotype when bred to homozygosity. Identified carrier frogs are currently being raised for phenotypic analysis.

The variable degree of mosaicism observed in F1 animals is not surprising, since there is evidence that the zygotic response to DNA damage is delayed in development (Stack and Newport, 1997; Ikegami et al., 1999). ENU-induced mutations that are 'fixed' during early cleavages will be less mosaic at a given locus than those fixed at later stages. Clearly, highly-mosaic loci can be more difficult to recover from F1 frozen sperm than less mosaic alleles such as the nfat mutation we have identified.

We circumvent this complication by generating the TILLING resource with adult non-mosaic F2 animals, and recovering carriers directly by mating the F2 individual in which the mutation was detected.

* quick link - http://q.sanger.ac.uk/mb7hsxp0