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First published online June 11, 2007
Journal of Experimental Biology 210, 2091-2103 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003186
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Specific endocytosis and degradation of naked DNA in the endocardial cells of cod (Gadus morhua L.)

Tore Seternes*, Tom C. Tonheim, Marie Løvoll, Jarl Bøgwald and Roy A. Dalmo

Department of Marine Biotechnology, The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway


Figure 1
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  		Fig. 1. Time course of the appearance of radioactivity in Atlantic cod tissues after intramuscular administration of trace amounts of 125I-fluorescein–pDNA (0.5–1 µg kg–1 body mass). The results are expressed as means + s.e.m. of five fish per time-point.

 

Figure 2
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  		Fig. 2. Time course of the appearance of radioactivity in Atlantic cod tissues after intravenous administration with trace amounts of 125I-fluorescein–pDNA (0.5–1 µg kg–1 body mass). The results are expressed as means + s.e.m. of five fish per time-point.

 

Figure 3
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  		Fig. 3. Stability of R70pRomiLuc in Atlantic cod blood after intravenous administration (A) and in vitro in whole blood (B). Supercoiled (SC) and open circular (OC) topoform were reduced to linear (L) topoform and degradation products, both in vitro and in vivo. M, molecular marker 1 kb plus (Invitrogen); N, negative control (blood without R70pRomiLuc); S, standard (R70pRomiLuc). The negative control demonstrates that there is no detectable endogenous pDNA in the blood.

 

Figure 4
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  		Fig. 4. Southern blot analysis of tissues 48 h after intramuscular administration of native pDNA (1 mg kg–1 body mass). SC, supercoiled topoform; OC, open-circular topoform,; L, linear topoform. All topoforms of pDNA (R70pRomiLuc) were detectable in all the investigated tissues, but degradation products were only visible in samples from the injection site

 

Figure 5
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  		Fig. 5. Fluorescence micrograph of atrium (A) and endocardium (B) of cod heart 24 h after intravenous injection of rhodamine–pDNA. The fluorescence can be seen in discrete vesicles evenly distributed throughout the cytoplasm of the endocardial cells.

 

Figure 6
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  		Fig. 6. Fluorescence micrograph of atrial endocardial endothelial cells (A) and cod head kidney leukocytes (B) cultured on glass coverslip and incubated with rhodamine–pDNA for 2 h. All cells in the monolayer cultures accumulated the pDNA. Note that fluorescence is confined to discrete vesicles which are probably endocytic vesicles.

 

Figure 7
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  		Fig. 7. Kinetics of endocytosis of 125I-fluorescein–pDNA in cultured cod atrial endocardial endothelial cells at 12°C (A) and head kidney leukocytes at 6°C (B). Monolayer cultures of aEEC or head kidney leukocytes in 2 cm2 wells were incubated with trace amounts of 125I-fluorescein–pDNA (approximately 2x104 c.p.m.; 20 ng). The results are presented as the cell-associated percentage of the total added radioactivity and are the mean of three independent experiments (aEEC). The result from the head kidney leucocyte study are the mean of cells from eight different fish.

 

Figure 8
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  		Fig. 8. Specificity of endocytosis of 125I-fluorescein–pDNA in cultured cod atrial endocardial endothelial cells. Monolayer cultures were incubated for 2 h at 12°C with trace amounts of labelled ligand (approximately 2x104 c.p.m.; 20 ng) alone (control) or together with excess amounts of unlabelled macromolecules (100 µg ml–1). The following macromolecules were used: plasmid DNA (pDNA), thymus DNA (ThDNA), formaldehyde-treated serum albumin (FSA), fucoidan and mannan. The results are presented as a percentage of the control value and are mean + s.e.m. of three independent experiments.

 

Figure 9
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  		Fig. 9. Southern blot analysis of degradation of pDNA in cultures cod atrial endocardial endothelial cells (aEEC). Monolayer cultures were incubated with 10 µg native pDNA and cells were solubilised after 1, 4, 24, 48 h. SC, super coiled topoform; OC, open-circular topoform; L, linear topoform. Open-circular and linear topoforms of pDNA (R70pRomiLuc) were detectable after 1 h. After 24 h, no intact pDNA was detected in the cells.

 




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