P. Collas et al., THE NUCLEAR-LOCALIZATION SEQUENCE OF THE SV40 TANTIGEN PROMOTES TRANSGENE UPTAKE AND EXPRESSION IN ZEBRAFISH EMBRYO NUCLEI, Transgenic research, 5(6), 1996, pp. 451-458
Citations number
27
Categorie Soggetti
Biology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
We report luciferase expression in zebrafish embryos after cytoplasmic
injection of low copy numbers of plasmid DNA coupled to the SV40 T an
tigen nuclear localization sequence (NLS). Binding of NLS to plasmid D
NA (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. Luciferas
e expression is induced in 35% of embryos with as low as 10(3) NLS-bou
nd pCMVL copies. With 10(4) copies, the proportion of expression incre
ases 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 freque
ncies of transgene expression without NLS. Regardless of the DNA conce
ntration, the use of NLS does not affect embryo viability for at least
up to 10 days. The specificity of NLS on luciferase expression was te
sted 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-b
ound 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 n
eed for using elevated DNA copy numbers in some gene transfer applicat
ions.