Nuclear localization signals enhance germline transmission of a transgene in zebrafish

We report that cytoplasmic injection into zebrafish eggs of 10(4) copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as co mpared to 10(6) copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration freq uency into the germline of founders. Monitoring the dynamics of nuclear upt ake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of in terphase nuclei indicates that NLS enhances both the proportion of nuclei i mporting DNA during early embryo development, and the amount of DNA importe d by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase ch ain reaction analysis of F(1)s. From germline transgenic DNA-NLS-injected f ounders, 47% transgenic F(1)s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injected founders (P < 0.01). In both cases, the tran sgene is transmitted to the F-2 generation. In addition, high-resolution FI SH analysis of transgenic F(1)s reveals that the use of NLS increases the n umber of distinct transgene integration sites along chromatin fibres.