Transfer, methylation and spontaneous mutation frequency of ΦX174am3cs70 sequences in medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus): Implications for gene transfer and environmental mutagenesis in aquatic species

This study describes the production of transgenic medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus) containing multiple copies of the bacteriophage ΦX174am3cs70. This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in ish and laboratory mammals. The ΦX174 sequence is unique in that there is no detectable homology with chromosomal DNA of medaka, mummichog or mice. The authors have compared cytoplasmic injection of 1–2 cell embryos with linear single copy and catenated constructs of the phage DNA. The catenated construct results in greater efficiency of gene transfer for both species in terms of copies per cell. Analyses of DNA from founder transgenic fish with methylation sensitive (HpaII) and methylation insensitive (MspI) restriction enzyme isoschizmers indicates CpG methylation of the integrated ΦX174 sequence. This study also demonstrates the efficient rescue of live phage from the chromosomal DNA of founder fish in sufficient numbers to determine a spontaneous mutation frequency for reversion of am3. A pooled sample of 20 μg DNA from four fish yielded 1.09 × 107 progeny phage with a spontaneous mutation frequency of 1.83 × 10−7. This spontaneous mutation frequency is similar to the spontaneous frequency for the same gene indictor recovered from transgenic mice. These results demonstrate that fish containing multiple copies of ΦX174 can be produced with no obvious detrimental effects and that the overall approach may be useful in basic and applied studies of environmental mutagenesis.