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 essential for effective cellular gene transfer applications. We report that noncovalent 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) cytoplasmically injected are sufficient to produce germline-transgenic zebrafish bearing a single copy of the transgene. This corresponds to a 10(5)-fold reduction 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 integration into the germline and somatic tissues is mosaic, and that the extent of mosaicism is negatively correlated with the amount of DNA-NLS injected. In addition, a larger proportion of zebrafish harboring a single copy of the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the potential use of nuclear targeting peptides noncovalently bound to vector DNA to enhance the efficiency of biotechnological nonviral gene transfer applications.
Liang
M R
MR
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway.
Aleström
P
P
Collas
P
P
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D015139:Blotting, Southern; D004247:DNA / Q000378:metabolism*; D015870:Gene Expression; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics*; D009030:Mosaicism; D019913:Nuclear Localization Signals / Q000502:physiology*; D019076:Transgenes / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*
D019913:Nuclear Localization Signals; D004247:DNA; D008156:Luciferases