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Fish Shellfish Immunol. 2008 Jan;24(1):90-101. Epub 2007 Oct 5.

Specific uptake of plasmid DNA without reporter gene expression in Atlantic salmon (Salmo salar L.) kidney after intramuscular administration.

Author information

1
Department of Marine Biotechnology, The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway. tom.tonheim@nfh.uit.no

Abstract

In this study we investigated tissue distribution of pDNA after intramuscular and intravenous administration, cellular localisation, receptor-specific uptake, integrity of pDNA and transgene expression in Atlantic salmon (Salmo salar L). Anatomical distribution of plasmid DNA was determined using both radiotracing and fluorescence microscopy. Cellular uptake was studied in cultures of adherent anterior kidney leucocytes. The integrity of the pDNA in vivo was investigated by Southern blot analysis. Transcription of plasmid DNA encoded luciferase gene and protein synthesis were investigated in salmon tissues by means of real-time reverse transcription-polymerase chain reaction and enzyme activity measurements, respectively. Approximately 50% of the total recovered radioactivity was redistributed from the carcass 168h after intramuscular administration and accumulated mainly in the kidneys (37% of total). The majority of radiolabelled plasmid DNA administered intravenously was taken up within the first 15min mainly by the kidney. Intravenous co-administration of trace amounts of radiolabelled plasmid DNA with excess amounts of unlabelled plasmid DNA or formaldehyde treated albumin (a ligand for the scavenger receptors) significantly inhibited accumulation of the radiotracer in the kidney. Fluorescence microscopy demonstrated that fluorescence was localised intracellularly in cells lining the sinusoids of the kidney after intravenous administration of rhodamine-labelled plasmid DNA. Southern blot analysis demonstrated presence of supercoiled plasmid DNA in all organs and tissue samples 168h after intramuscular administration, but degradation products were only revealed at the administration site. Luciferase transcript and activity were only detectable at the administration site 24-168h after intramuscular administration of plasmid DNA. After incubation with trace amounts of radiolabelled plasmid DNA, only minor amounts of radiolabelled plasmid DNA were cell associated in cultures of adherent anterior kidney leucocytes. These results suggested that a substantial portion of radiolabelled plasmid DNA was redistributed from the carcass and was mainly cleared by a receptor-specific uptake in the kidney. Although intact plasmid DNA was detected in the kidney and other tissues, no luciferase transcripts or activity were detected in these samples at any time points investigated (24-168h), except for the administration site following intramuscular administration.

PMID:
18023591
DOI:
10.1016/j.fsi.2007.09.006
[Indexed for MEDLINE]

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