The SV40 T antigen nuclear localization sequence enhances nuclear import of vector DNA in embryos of a crustacean (Litopenaeus schmitti).
A genetic transformation system for penaeid shrimp could provide a powerful technique for the improvement of different production traits of importance for a sustainable aquaculture. The development of a successful transformation system depends on the ability to efficiently introduce exogenous DNA into the target species. The ability of the nuclear localization signal (NLS) peptide of the SV40 T antigen to facilitate nuclear import and transient gene expression is known from vertebrate systems and for the first time, is shown here to be efficient in a crustacean species, i.e. the shrimp Litopenaeus schmitti. Electroporation was used to introduce the pCMV-lacZ plasmid that contains the human cytomegalovirus promoter/enhancer (CMV) fused to the beta-galactosidase (lacZ) coding region, into L. schmitti zygotes. Supercoiled DNA was used at 50 or 500 ng/microl naked or bound to NLS peptide. The hatching rate of electroporated zygotes was around 60% for all groups, except from the pCMV-lacZ:NLS group at 500 ng/microl (43%). Based on Southern blot analyses of polymerase chain reaction (PCR) products the gene transfer frequency was 2-fold higher using DNA:NLS complexes than with naked DNA (23.8% vs. 11.5%, with 50 ng/microl of plasmid DNA, 44.3% vs. 28.8% with 500 ng/microl). The beta-galactosidase activity assay indicated that nuclear uptake is faster for the DNA:NLS complexes than for naked DNA. The beta-galactosidase activity was always higher in the DNA:NLS groups than in the naked DNA groups. To our knowledge, this is the first report on the use of an NLS peptide to improve gene transfer and nuclear uptake in crustaceans.
Center of Genetic Engineering and Biotechnology, P.O. Box 387, ZIP 70100 Camagüey 1, Cuba. Amilcar.Arenal@cigb.edu.cu
Amilcar
A
Arenal
Rafael
R
Pimentel
Carmen
C
García
Eulogio
E
Pimentel
Peter
P
Aleström
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism*; D047108:Embryonic Development; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics*; D010063:Ovum / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D033561:Penaeidae / Q000196:embryology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D019913:Nuclear Localization Signals; D004247:DNA; D001616:beta-Galactosidase