The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei |
| |
Authors: | Philippe Collas Harald Husebye Peter Aleström |
| |
Affiliation: | (1) Department of Biochemistry, Norwegian College of Veterinary Medicine, PO Box 8146 Dep., 0033 Oslo, Norway;(2) Department of Food Science, Agricultural University of Norway, PO Box 5036, 1432 As, Norway |
| |
Abstract: | 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 0.25m 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 103 NLS-bound pCMVL copies. With 104 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 NLS-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. |
| |
Keywords: | luciferase activity transient expression microinjection nuclear localization signal Danio rerio |
本文献已被 SpringerLink 等数据库收录! |
|