Glowing zebrafish: Integration, transmission, and expression of a single luciferase transgene promoted by noncovalent DNA-nuclear transport peptide complexes

The development of vehicles driving foreign DNA into the cell nucleus is es sential for effective cellular gene transfer applications. We report that n oncovalent binding of nuclear localization signal (NLS) peptides to plasmid DNA enhances nuclear uptake of the DNA and promotes germline integration, inheritance, and expression of a single copy of a luciferase reporter gene in zebrafish. As few as 10 DNA-NLS complexes (0.06 fg plasmid DNA) cytoplas mically injected are sufficient to produce germline-transgenic zebrafish be aring a single copy of the transgene. This corresponds to a 10(5)-fold redu ction in DNA concentration compared to commonly used procedures. Use of 10( 3) or 10(4) DNA-NLS complexes augments the number of transgene integrations , which occur mostly within 1-4 distinct insertion sites in the genome. In situ hybridization analyses and transmission studies show that transgene in tegration into the germline and somatic tissues is mosaic, and that the ext ent of mosaicism is negatively correlated with the amount of DNA-NLS inject ed. in addition, a larger proportion of zebrafish harboring a single copy o f the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the po tential use of nuclear targeting peptides noncovalently bound to Vector DNA to enhance the efficiency of biotechnological nonviral gene transfer appli cations. (C) 2000 Wiley-Liss, Inc.