spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

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
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seternes, T.
Right arrow Articles by Dalmo, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seternes, T.
Right arrow Articles by Dalmo, R. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

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
View larger version (7K):
[in this window]
[in a new window]

  		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
View larger version (12K):
[in this window]
[in a new window]

  		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
View larger version (21K):
[in this window]
[in a new window]

  		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
View larger version (18K):
[in this window]
[in a new window]

  		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
View larger version (66K):
[in this window]
[in a new window]

  		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
View larger version (52K):
[in this window]
[in a new window]

  		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
View larger version (7K):
[in this window]
[in a new window]

  		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
View larger version (5K):
[in this window]
[in a new window]

  		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
View larger version (15K):
[in this window]
[in a new window]

  		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.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007