The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei.
We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25 M KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 10(3) NLS-bound pCMVL copies. With 10(4) copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p < 0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days. The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of MLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.
Collas
P
P
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway.
Husebye
H
H
Aleström
P
P
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology; D004627:Embryonic Induction; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005818:Genetic Engineering / Q000379:methods / Q000706:statistics & numerical data; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008845:Microinjections; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D010446:Peptide Fragments; D008156:Luciferases