Xenopus tropicalis Mutation Resource

The Mutant Library

Our groups have assessed the efficiency of both gamma-ray induced mutation, and chemically induced mutation. We concentrate here on the induction of mutations by ENU, since this is ideally suited to the identification of simple mutations by resequencing. We performed a pilot study for the recovery of chemically induced mutations. In the study we screened both for induced mutant phenotypes, in a forward screen, and for heterozygous changes using a reverse genetic approach generally known as TILLING (McCallum et al., 2000 [10748531], Greene et al., 2003 [12807792], Till et al., 2003 [14501067], Wienholds et al., 2003 [14613981], Winkler et al., 2005 [15867432]).

Typically to calibrate the appropriate level of chemical mutagenesis, one would use homozygous viable mutant pigmentation alleles. By fertilizing eggs of heterozygous females with mutagenized sperm, or sperm from mutagenized males, an induced mutation rate can be estimated from the number of pigmentation mutants to arise from the cross. For example, in zebrafish albino or golden loci are often used to characterize the induced mutation rate (Grunwald and Streisinger, 1992 [1628817], Mullins et al., 1994 [7922324], Solnica-Krezel et al., 1994 [8013916], Riley and Grunwald et al., 1995 [7597068]). Unfortunately, there were not any such mutant alleles available for Xenopus tropicalis when we started; we therefore opted to use a mutagenesis method we thought had the best chance of inducing the most mutations possible. Specifically, we mutagenized mature sperm in vitro followed by in vitro fertilization of eggs to produce populations of F1 carriers (Grunwald and Streisinger, 1992 [1628817], Riley and Grunwald et al., 1995 [7597068]). Because mature sperm were being used and each strand of each haploid chromosome is independently modified by ENU, F1 carriers are genetic mosaics, with different mutations being fixed in each blastomere after the first cleavage.

Mature F1 females were screened by gynogenetic methods to identify induced phenotypes (forward screen, see Phenotyping protocol) and both males and females were used to produce populations of non-mosaic F2 frogs, which were then screened for induced mutations in specific genes (reverse screen, see Tilling protocol).

During early stages of development of F1 embryos effects of excessive mutagenesis can be seen as a significant increase in the incidence of malformation and a high rate of embryonic lethality. We measured lethality at neurula stages as a function of the ENU concentration to which sperm were exposed (Table and Figure). We found that at relatively low doses of ENU (1 mM) (data not shown) the incidence of lethality and malformation was comparable to that of untreated sperm, whereas 5 mM, 10 mM and 15 mM ENU treatments produced significant defects and lethality among F1 neurulae. These results are consistent with a production of dominant and/or synthetic lethal mutations.

ENU concentration Dead Gastrulation Defects Other Defects Normal Total
0 mM 1 (1%) 8 (10%) 1 (1%) 68 (87%) 78
5 mM 4 (3%) 24 (16%) 8 (5%) 115 (76%) 151
10 mM 6 (4%) 40 (26%) 20 (13%) 85 (56%) 151
15 mM 5 (10%) 20 (42%) 9 (19%) 14 (29%) 48
Figure 1.

Figure 1.


Because the F1 animals are mosaic, we measured the induced mutation rate in families of F2 progeny from a group of 63 F1 adult males. With the intention of screening the spectrum of mutations present in the 63 F1 parents we aimed to collect 24 F2 tadpoles per parent and ultimately generated a library of 1,395 F2 tadpoles. Genomic DNA was recovered from 5-day-old tadpoles, amplified by nested PCR and screened by direct sequencing of amplicons. To bias towards detection of nonsense mutations giving loss of function phenotypes, primers were designed to amplify 150-350 bp of amino-terminal coding exons. Forward sequence was generated for each of the 1,395 PCR products for each of the amplicons, and processed by a Mutation Finder program (Bosman et al, 2005 [16207732]; Goda et al, 2006 [16789825]) that compares each trace to a reference sequence and identifies potential mutations in their heterozygous state.

We employed this approach to obtain a molecular mutation rate for the 10mM ENU treated population. In a duplicate experiment screening five amplicons, we confirmed 24 base changes in approximately 1 MB of sequence (see Tilling Pipeline). Using these data, we estimate a mutation rate of 1 base change every 98,624 ± 49,983 bases. From the results of the Mutant Resource Project on zebrafish we can estimate the rate of mutation recovery for X. tropicalis. e have found that one nonsense mutation occurs for every 20 mutations identified. With a rate of 1 mutation for every 100,000 analyzed bases, we would need to sequence 2 Mb on average to find a single nonsense mutation. With the proposed library of 6000 non-mosaic F2 individuals and an average amplicon sequence read of 700 bp from each individual, we have more than 4Mb, or an average of more than two nonsense alleles for each gene analyzed.


  • Genetic screens for mutations affecting development of Xenopus tropicalis.

    Goda T, Abu-Daya A, Carruthers S, Clark MD, Stemple DL and Zimmerman LB

    PLoS genetics 2006;2;6;e91

  • Multiple mutations in mouse Chd7 provide models for CHARGE syndrome.

    Bosman EA, Penn AC, Ambrose JC, Kettleborough R, Stemple DL and Steel KP

    Human molecular genetics 2005;14;22;3463-76

  • Target-selected mutant screen by TILLING in Drosophila.

    Winkler S, Schwabedissen A, Backasch D, Bökel C, Seidel C, Bönisch S, Fürthauer M, Kuhrs A, Cobreros L, Brand M and González-Gaitán M

    Genome research 2005;15;5;718-23

  • Efficient target-selected mutagenesis in zebrafish.

    Wienholds E, van Eeden F, Kosters M, Mudde J, Plasterk RH and Cuppen E

    Genome research 2003;13;12;2700-7

  • Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.

    Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L and Henikoff S

    Genetics 2003;164;2;731-40

  • High-throughput TILLING for functional genomics.

    Till BJ, Colbert T, Tompa R, Enns LC, Codomo CA, Johnson JE, Reynolds SH, Henikoff JG, Greene EA, Steine MN, Comai L and Henikoff S

    Methods in molecular biology (Clifton, N.J.) 2003;236;205-20

  • Targeted screening for induced mutations.

    McCallum CM, Comai L, Greene EA and Henikoff S

    Nature biotechnology 2000;18;4;455-7

  • Efficient induction of point mutations allowing recovery of specific locus mutations in zebrafish.

    Riley BB and Grunwald DJ

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;13;5997-6001

  • Efficient recovery of ENU-induced mutations from the zebrafish germline.

    Solnica-Krezel L, Schier AF and Driever W

    Genetics 1994;136;4;1401-20

  • Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate.

    Mullins MC, Hammerschmidt M, Haffter P and Nüsslein-Volhard C

    Current biology : CB 1994;4;3;189-202

  • Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea.

    Grunwald DJ and Streisinger G

    Genetical research 1992;59;2;103-16

* quick link - http://q.sanger.ac.uk/ydmu0icx