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 compared to 10(6) copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported 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 chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injected founders (P < 0.01). In both cases, the transgene is transmitted to the F2 generation. In addition, high-resolution FISH analysis of transgenic F1s reveals that the use of NLS increases the number of distinct transgene integration sites along chromatin fibres.
Collas
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P
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway.
Aleström
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D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D005260:Female; D017404:In Situ Hybridization, Fluorescence; D019913:Nuclear Localization Signals / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*
D017931:DNA Primers; D004274:DNA, Recombinant; D019913:Nuclear Localization Signals