TRANSFER, METHYLATION AND SPONTANEOUS MUTATION FREQUENCY OF PHI-X174AM3CS70 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 lati pes) and mummichog (Fundulus heteroclitus) containing multiple copies of the bacteriophage Phi X174am3cs70. This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in fish and laboratory mammals, The Phi X174 sequence is uni que in that there is no detectable homology with chromosomal DNA of me daka, mummichog or mice. The authors have compared cytoplasmic injecti on of 1-2 cell embryos with linear single copy and catenated construct s of the phage DNA. The catenated construct results in greater efficie ncy of gene transfer for both species in terms of copies per cell, Ana lyses of DNA from founder transgenic fish with methylation sensitive ( HpaII) and methylation insensitive (MspI) restriction enzyme isoschizm ers indicates CpG methylation of the integrated Phi X174 sequence, Thi s study also demonstrates the efficient rescue of live phage from the chromosomal DNA of founder fish in sufficient numbers to determine a s pontaneous mutation frequency for reversion of am3. A pooled sample of 20 mu g DNA from four fish yielded 1.09 x 10(7) progeny phage with a spontaneous mutation frequency of 1.83 x 10(-7). This spontaneous muta tion frequency is similar to the spontaneous frequency for the same ge ne indictor recovered from transgenic mice. These results demonstrate that fish containing multiple copies of Phi X174 can be produced with no obvious detrimental effects and that the overall approach may be us eful in basic and applied studies of environmental mutagenesis.