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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
Specific endocytosis and degradation of naked DNA in the endocardial cells of cod (Gadus morhua L.)
Department of Marine Biotechnology, The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway
* Author for correspondence (e-mail: tore.seternes{at}nfh.uit.no)
Accepted 2 April 2007
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125I-labelled pDNA was rapidly eliminated from the blood by the aEEC of the cod heart atrium and ventricle. Co-injection of trace amounts of 125I-labelled pDNA with excess amounts of non-labelled pDNA or formaldehyde-treated albumin (FSA), a ligand for the cod EEC scavenger receptor, significantly inhibited the accumulation of the radiotracer in the heart. The organ to blood ratio of radioactivity after inhibition of the cod EEC scavenger receptor demonstrated that the radioactivity not taken up by the EEC remained in the blood. Fluorescence microscopy of tissue sections from cod injected with fluorescein-labelled pDNA confirmed intracellular uptake of pDNA by the endocardial cells of the atrium and ventricle. In purified cultures of cod aEEC the fluorescein-labelled pDNA was taken up in structures reminiscent of endosomal/lysosomal vesicles. Uptake of 125I-labelled pDNA in cultures of cod aEEC was specific. Incubation of cultures with 125I-labelled pDNA together with excess amounts of FSA and fucoidan, which are molecules also known to bind to the scavenger receptors, reduced the uptake of the pDNA by at least 70%. Mannan, a ligand for the mannose receptor, did not inhibit the uptake of 125I-labelled pDNA. Despite, low uptake of 125I-fluorescein-pDNA in the kidney of the cod, the uptake of pDNA in cultured cod head kidney leukocytes was significant.
Southern blot analysis of cod tissues after injection of pDNA and culture of aEEC given 10 µg pDNA per 106 cells demonstrated the presence of degradation products in tissues and in the cell cultures. Real-time RT–PCR studies showed expression of luciferase mRNA only at the injection site 168 h after injection. Neither expression of luciferase mRNA nor luciferase activity was present in cod aEEC incubated for 48 h with 10 µg pDNA.
These results suggest that the EEC are very important for removal of blood borne pDNA in cod and that the uptake by these cells was mediated in a scavenger–receptor-like manner. Uptake of pDNA by head kidney leukocytes was only observed in vitro. The endocytosed DNA was subjected to intracellular degradation and was not expressed by the cod EEC. Despite the low amount of radioactivity found in the head kidney after i.v. injection of 125I-labelled pDNA, the head kidney leukocytes seem to have a high capacity for uptake of 125I-labelled pDNA in vitro.
Key words: DNA, scavenger receptor, endocytosis, endothelium, endocardium, Atlantic cod, Gadus morhua
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). A DNA vaccine
consists of a bacterial plasmid with a strong viral promoter, the gene(s) of
interest and a polyadenylation and transcriptional termination sequence. The
plasmid is grown in bacteria, purified, dissolved in a saline solution, and
then administered by intramuscular (i.m.) injection of naked DNA (ng and µg
amounts) to activate protein expression in vivo and to ultimately
induce an immune response and disease protection. Studies with reporter genes
have demonstrated that fish cells efficiently express foreign proteins encoded
by non-viral eukaryotic expression vectors
(Anderson et al., 1996b;
Kanellos et al., 1999).
Intramuscular injection of a plasmid encoding the glycoprotein of the
infectious haematopoietic necrosis virus (IHNV) and the G-protein of viral
hemorrhagic septicaemia virus (VHS) have been reported to induce protection
against experimental challenge in rainbow trout and in sockeye and Chinook
salmon fry (Corbeil et al.,
1999; Garver et al.,
2005; Traxler et al.,
1999), and to induce antibody production
(Anderson et al., 1996a). In
Canada, an IHNV DNA vaccine (Apex-IHN®) developed by Aqua Health, Ltd, an
affiliate of Novartis, was cleared for marketing by the Canadian Food
Inspection Agency on July 15 2005 (Novartis media release July 19, 2005). It
is highly likely that this is the first of several DNA-based vaccines for
aquacultured fish. A prerequisite for production of a transgene is that the
DNA vaccine (naked pDNA) is taken up by the cells, transferred to the cytosol
and eventually transported to the nucleus before any expression occurs.
Several passive and active mechanisms have been described for receptor binding
and/or uptake of DNA. The uptake processes are different forms of endocytosis:
phagocytosis (cell eating) and pinocytosis (cell drinking). Pinocytosis is
utilised by essentially all cell types and occurs by multiple pathways, i.e.
clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin- and
caveolin-independent endocytosis and macropinocytosis
(Belting et al., 2005).
Macrophages, granulocytes and dendritic cells carry out phagocytosis (e.g.
dead cells, bacteria, large molecular complexes), and they are found in many
parts of an animal. In particular, those macrophages residing in close
connection to the bloodstream, are highly phagocytic thus functioning as an
important element in the reticuloendothelial system – together with
scavenger endothelial cells in liver of mammals and in the kidney or heart of
pisciformes (fish) (Seternes et al.,
2002). The endocytic pathway normally confers total degradation of
DNA – especially if the DNA is transported to the end-point terminal
(lysosomes). However, tiny amounts of (non-protected) DNA may escape the
endocytic compartments and degradation. This DNA may be trafficked through the
nuclear membrane into the nucleus where transcription occurs.
The endocardial endothelial cells (EEC) lining the muscular trabecula of
the Atlantic cod (Gadus morhua L.) heart represent a general
vertebrate non-phagocytic scavenger endothelial cell (SEC) system with an
extensive capacity to endocytose and degrade soluble physiological and foreign
macromolecular waste substances/molecules from the circulation by
receptor-mediated endocytosis (Seternes et
al., 2002). Functional studies indicate that the cod aEECs express
a set of at least four types of functional endocytic receptors for this
purpose: (i) the collagen 
chain receptor
(Koren et al., 1997;
Smedsrød et al., 1995),
(ii) the hyaluronan receptor (Seternes et
al., 2001b; Sørensen et
al., 1997a), (iii) the mannose receptor
(Sørensen et al., 2001)
and (iv) the scavenger receptor (Seternes
et al., 2001a; Sørensen
et al., 1998).
Several studies in mammals have shown that the liver is the main organ
responsible for rapid clearance of DNA from the circulation
(Emlen and Mannik, 1978;
Emlen and Mannik, 1984;
Gauthier et al., 1996). In the
rat liver the scavenger endothelial cells rather than Kupffer cells have been
shown to be responsible for the highest uptake and degradation of pDNA
(Hisazumi et al., 2004).
Whether fish scavenger endothelial cells are active in uptake and degradation
of pDNA is at present not known. The injected pDNA vaccine is subjected to
many hurdles before any expression of the transgene occurs. The first being
mucosal-derived and blood plasma nucleases
(Kawabata et al., 1995), the
second hurdle is cellular degradation
(Odaka and Mizuochi, 1999),
the third is cytosolic degradation of DNA vaccine, and the process of nuclear
transfer and localisation of pDNA/DNA fragments represents an obstacle for
transgene expression (Lechardeur et al.,
1999). The aim of this study was to examine the tissue
distribution of pDNA assisted by recepto-specific uptake, transgene expression
and persistence of pDNA in Atlantic cod – a fish species emerging as
highly interesting for aquaculture and a fish species that have been shown to
be susceptible to several viral diseases
(Samuelsen et al., 2006).
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Animals
Fish for in vivo studies, Atlantic cod (Gadus morhua L.;
50–100 g) were obtained from Tromsø Aquaculture station
(Norwegian Institute of Fisheries and Aquaculture Research, Tromsø,
Norway). The fish were kept in plastic tanks (2000 litre) supplied with
running seawater at 4°C. All fish were adapted to the test conditions for
at least 1 week before the experiments started and fed a commercial diet daily
before and during the adaptation and experimental period. For cell isolation,
hearts from wild captured Atlantic cod (1–4 kg) were used. The fish were
kept in plastic tanks supplied with running seawater (4°C) and fed a
commercial diet. The experiments reported in this manuscript were conducted in
accordance with The European Convention for the Protection of Vertebrate
Animals used for Experimental and other Scientific Purposes of 18 March
1986.
Plasmids and ligands for endocytosis studies
The R70pRomiLuc plasmid contained a firefly (Phontinus pyralis)
luciferase gene under the control of a murine cytomegalovirus immediate early
promoter (CMV-IEP), a generous gift from Dr Üwe Fischer (Federal Research
Centre for Virus Disease of Animals, Germany). Plasmid DNA was propagated by
transformations of Escherichia coli DH5 grown in Luria Bertani
medium supplied with 100 µg ml–1 ampicillin and isolated
using Qiagen Plasmid Mega Kit (Qiagen, GmbH, Hilden, Germany) according to the
manufacturer's recommendation. Purity and concentration of pDNA were measured
using a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies,
Wilmington, DE, USA), and A260/A280 ratios between 1.8
and 2.0 were considered pure. Gel electrophoresis analysis confirmed that all
pDNA preparations were free of detectable genomic DNA and RNA contamination.
Expected endotoxin content was given as 9.3 EU µg–1
mg–1 purified pDNA by the manufacturer. Mannan and fucoidan
were purchased from Sigma Chemical Co.
Preparation of fluorescein-labelled pDNA
The nucleic acid labelling kit LabelIT-fluorescein (MIR 3200) and
LabelIT-TM-Rhodamine (MIR 4100) was obtained from Mirus Corp.
(Madison, WI, USA). Synthesis of the LabelIT reagents is described in
US Patent US06262252. Plasmid DNA was modified using LabelIT reagents
according to the manufacturer's recommendations. Briefly, LabelIT
reagent and pDNA were combined in 10x labelling buffer A at 0.5:1 (v:w)
ratio of LabelIT reagent to pDNA and incubated at 37°C for 1 h.
The labelled pDNA was separated from unattached label using G50 Microspin
purification columns (Amersham Bioscience, Buckinghamshire, UK).
Fluorescein–pDNA and rhodamine–pDNA were digested with DNase
(TURBO DNA-free; Ambion, Austin, TX, USA) to verify the attachment of
fluorescein to the pDNA. Plasmid DNA and fluorescein–pDNA and
rhodamine–pDNA were then run on 0.7% agarose gels.
Preparation of 125I-labelled pDNA
The fluorescein–pDNA was radiolabelled with carrier-free
Na[125I] in a direct reaction using 1,3,4,6-tetrachloro-3,
6-diphenylglycoluril (Iodogen; Pierce, Rockford, IL, USA) as oxidising
agent, according to the manufacturer's instructions. Free iodine was removed
from the solution by gel filtration on a PD-10 column eluted with
phosphate-buffered saline (PBS). The specific activity of
125I-fluorescein–pDNA was determined to be
1.1x106 c.p.m. µg–1. Radioactivity was
measured using a Packard gamma counter (Packard Instrument Company, Meriden,
IL, USA). Radiolabelled fluorescein–pDNA was digested with DNase
(Ambion) or proteinase K (Qiagen) to verify the attachment of radiolabel to
the fluorescein–pDNA. To investigate if any free active fluorescein
label was still present, human serum albumin (HSA) was added to the
radiolabelled fluorescein–pDNA before running the samples on a 0.7%
agarose gel and visualisation on a Hyperfilm-HP (Amersham Bioscience).
Transfection efficiency of native and modified pDNA
To ensure that the luciferase gene could still be correctly expressed after
labelling pDNA with fluorescein, 0.4 µg of labelled or unlabelled plasmid
was transfected into semiconfluent Caco-2 cells in the presence of
Lipofectamine Plus according to the manufacturer's instructions (Invitrogen,
Oslo, Norway). In short, approximately 2.7x104 cells were
seeded per well in 24-well plates and cultured at 37°C in 5%
CO2 for 24 h to obtain 80% confluence in Eagle's minimum essential
medium (EMEM) supplemented with 20% foetal bovine serum (FBS). Plasmid DNA
(0.4 µg), 4 µl Plus reagent and 1.5 µl Lipofectamine reagent were
added to each well and incubated at 37°C for 3 h. The transfection medium
was replaced with growth medium. After 48 h of incubation the monolayers of
cells were harvested for luciferase assay. Luciferase activity was assayed
according to manufacturer's protocol using a Luminoskan Ascent® microplate
illuminometer (Thermo Electron Oy, Vantaa, Finland). The relative light units
were normalised to the protein concentrations in the samples determined by the
Bio-Rad RC DC protein assay (Bio-Rad Laboratories).
Anatomical distribution
Atlantic cod (50–100 g) were anaesthetised by immersion in 0.004%
(w/v) metacaine solution and injected intravenously (35 fish) or
intramuscularly (25 fish) with trace amounts of
125I-fluorescein–pDNA (0.5–1 µg
kg–1 body mass) in a total injection volume of 100 µl PBS
per fish for the i.v. injection and 100 µl PBS per fish for the i.m.
injection. At specified time intervals, blood samples were collected from the
caudal vein approximately 2 cm caudal to the injection site of anaesthetised
fish. The fish were killed by a blow to the head immediately after blood
sampling, and the heart, anterior kidney, spleen, liver, intestine and blood
were removed and analysed for radioactivity. The carcass of the fish,
including head, gills, muscle tissue and skin were put in one tube and
analysed for radioactivity. Parallel series of five fish were collected 5 min,
15 min 1, 4, 24, 48 and 168 h after intravenous (i.v.) injection, and 1, 4,
24, 48 and 168 h after i.m. injection. Total blood mass was set to 5% of body
mass (Skov and Steffensen,
2003).
Inhibition studies were performed by co-injecting i.v. trace amounts of 125I-fluorescein–pDNA (0.5–1 µg kg–1 body mass) with unlabelled pDNA (3.2 mg kg–1 body mass) or formaldehyde-treated albumin (FSA; 5–10 mg kg–1 body mass) in a total injection volume of 100 µl PBS per fish. A total of 15 cod were used in this experiment, five were injected with 125I-fluorescein–pDNA/pDNA, five with 125I-fluorescein–pDNA/FSA and five with 125I-fluorescein–pDNA only. After 1 h, blood samples were collected from the caudal vein approximately 2 cm caudal to the injection site. The fish were killed with a blow to the head immediately after blood sampling. The heart, anterior kidney, liver and blood were analysed for radioactivity.
Localisation of radiolabelled pDNA in blood
Uptake of pDNA by blood cells was investigated by density gradient
sedimentation of whole blood after i.v. and i.m. injection of
125I-labelled pDNA. A total of 10 cod were anaesthetised and
injected i.v. (5 fish) and i.m. (5 fish) with
125I-fluorescein–pDNA (0.5-1 µg kg–1 body
mass) in a total injection volume of 100 µl PBS per fish. Blood samples
were collected in heparin-coated tubes 1 h and 24 h after injection. A
discontinuous gradient was made up of 10 ml 25% and 30 ml 50% Percoll®
density medium. Whole blood (2 ml) diluted in 8 ml of L15 cell culture medium
supplemented with heparin (50 i.u. ml–1) were loaded onto
each density gradient for centrifugation at 400 g for 40 min
at 4°C in a centrifuge equipped with a swing-out rotor (Kubota 8800,
Kubota Corporation, Tokyo, Japan). After centrifugation, 10 fractions of 5 ml
were collected from the gradients. Fractions 1–4 contained plasma,
fractions 5–7 contained the 25–50% interface where leukocytes are
trapped, and fractions 7–10 contained red blood cells. The fractions
were analysed for radioactivity.
In vivo and in vitro stability of pDNA in blood
The stability of pDNA in blood of Atlantic cod was assayed at 4°C
in vivo and in vitro by gel electrophoresis. A total of five
fish were injected i.v. with pDNA (5 mg kg–1 body mass).
Blood samples were collected 1, 5, 15, 30 and 60 min post-injection in
EDTA-coated syringes. For the in vitro study, blood was collected
from five fish in heparin-coated tubes and mixed with pDNA to a final
concentration of 50 µg ml–1 blood, corresponding to the
calculated initial blood concentration after i.v. injection. The pDNA
containing blood was incubated for 1, 5, 15, 30 and 60 min. The blood was then
immediately mixed with the deoxyribonuclease inhibitor EDTA to a final
concentration of 25 mmol l–1 to stop pDNA degradation.
Plasmid DNA was extracted from the plasma with equal volumes of
phenol/chloroform/isoamyl alcohol (25:24:1, w/v; Invitrogen). The aqueous
phase containing pDNA was ethanol precipitated and 500 ng of DNA applied to a
0.7% agarose gel for electrophoresis, stained with ethidium bromide and
visualised in a transilluminator.
Histological preparations
Rhodamine–pDNA was detected by fluorescence microscopy 1 h after i.v.
injection of pDNA (0.5 mg kg–1 body mass) into Atlantic cod.
The fish were killed by a blow to the head, and the heart, anterior kidney,
spleen and liver were dissected out and fixed in 4% formaldehyde. Paraffin
sections were prepared and then dewaxed in xylene and mounted in Histokitt.
Specific fluorescence due to rhodamine–pDNA was observed as a bright red
colour. Sections were examined using a Leica photomicroscope equipped with
incident-light fluorescence optics. Pictures were taken with a Leica digital
camera (Leica Microsystems, Wetslar, Germany).
Isolation and cultivation of atrial endocardial endothelial cells
Functionally intact atrial endocardial endothelial cells (aEEC) from cod
were purified according to (Koren et al.,
1997). In short, the heart was dissected out and perfused with
L-15 medium supplied with heparin (10 i.u. ml–1). The atria
were dissected free and cut open. Ostial tissue rich fibrocytes and
macrophages were discarded before transfer to a 50 ml sterile plastic tube
with 25 ml of calcium-free buffer (Pertoft
and Smedsrod, 1987). After 30 min incubation with horizontal
shaking (250 cycles min–1) the buffer was changed. The atria
were then incubated with the following solutions: (i) trypsin (0.5 mg
ml–1) and EDTA (0.1 mg ml–1) in PBS for 5
min; (ii) collagenase (0.5 mg ml–1 in L-15 medium
supplemented with 0.7 mg ml–1 CaCl2
2H2O) for 30 min. The contents of the tube were transferred to a
sterile Petri dish, and the atrium was flushed several times with the jet from
a 10 ml plastic syringe, re-using the obtained cell suspension. The remaining
tissue was discarded and the cell suspension was centrifuged for 5 min at 400
g, and the pellet was resuspended in L-15. Contaminating
macrophages (adherent cells) were removed according to the method of
Sørensen et al. (Sørensen et
al., 1998). The non-adherent cells were seeded on plastic (Falcon,
Becton Dickinson & Company, NJ, USA) or glass tissue culture slides
precoated with fibronectin (0.5 mg ml–1). The incubation
medium was L-15 supplemented with 10% FBS. The isolation procedure and
incubations were carried out at 12–14°C. The cells were washed with
L-15 medium after 24 h and used in experiments the same or the next day. The
number of cells seeded per 2 cm2 was approximately 106.
Contamination by cardiomyocytes and a few macrophages were observed. On
average, more than 90% of the cells were aEEC, as evaluated by morphological
characteristics.
Isolation and cultivation of cod head kidney macrophages
Cultures of head kidney macrophages were established as described
previously (Braun-Nesje et al.,
1981), with some modification by
(Sørensen et al.,
1997b). Briefly, the bilobular head kidney tissue was aseptically
removed by ventral incision and minced through a 70 µm sterile nylon cell
strainer (Falcon®; Becton & Dickinson Company, NJ, USA) with 10 ml
L-15 medium containing heparin. A discontinuous gradient was made up of 10 ml
25% and 30 ml 51% Percoll solutions and no more than 10 ml of the cell
suspension were loaded onto each density gradient for centrifugation at 400
g for 40 min at 4°C. The cells banding in the 25–51%
interface were collected for cultivation of macrophages. Aliquots of the cell
suspension, containing 1–5x106 cells
ml–1, were plated in dishes for endocytosis studies.
Endocytosis of radiolabelled pDNA by cultured cod aEEC and cod head kidney leukocytes
AEEC and head kidney leukocytes cultures established in 2 cm2
wells (approximately 3x105 cells attached and spread per
cm2) were washed three times with L-15 medium and supplied with
fresh medium containing 1% HSA and trace amounts of
125I-fluorescein–pDNA (20 000 c.p.m.) in a total incubation
volume of 200 µl per well. Incubations of
125I-fluorescein–pDNA at 12°C were terminated after
various times by removing incubation medium along with one washing volume of
500 µl PBS. Cell-associated ligand was quantified by solubilising the cell
layer with 1% SDS, followed by counting in a gamma counter. Receptor-specific
endocytosis of 125I-fluorescein–pDNA was examined in cod aEEC
by inhibition studies. Monolayer cultures were incubated for 2 h with trace
amounts of labelled ligand alone (control) or together with excess amounts of
non-labelled macromolecules (100 µg ml–1). Endocytosis
experiments were terminated after 2 h at 12–14°C by removal of
incubation medium and one washing volume of 500 µl PBS to a PD-10 column.
Intact ligand was eluted in the void volume and degraded ligand in the total
volume. Cell-associated ligand was quantified by solubilising the cell layer
with 1% SDS, followed by counting in a gamma counter.
Fluorescence microscopy of cod aEEC after incubation with rhodamine–pDNA
Cultures of aEEC were established on glass coverslips. Rhodamine–pDNA
(10–50 µg ml–1) was incubated together with the
cultures for 2 h at 12–14°C. The cultures were fixed in 4%
formaldehyde and embedded in an antifading medium (Dako fluorescent mounting
medium, Glostrup, Denmark). The specific fluorescence due to
Rhodamine–pDNA was observed as a bright red colour. Sections were
examined using a Leica photomicroscope equipped with incident-light
fluorescence optics. Pictures were taken using a Leica digital camera.
Gene transfer
The pDNA (1.0 mg kg–1 body mass) was injected i.m. into
Atlantic cod in an injection volume of 100 µl PBS. Parallel series of three
fish were collected 24, 48 and 168 h after injection. The fish were killed
with a blow to the head, and heart, kidney, liver, spleen and injection site
were aseptically removed. Blood samples were taken from the caudal vein, with
EDTA as an anticoagulant. Tissues harvested for Southern blot analysis,
real-time reverse transcription polymerase chain reaction (RT–PCR) and
luciferase assay were put directly into liquid nitrogen, then kept at
–86°C.
Monolayer cultures of aEECs (pooled cells from three fish, approximately 3x105 cells cm2) were established in 9.6 cm2 dishes. The cells were incubated with 10 µg pDNA in a total volume of 600 µl L-15 medium supplemented with 1% HSA. Incubation of pDNA at 12°C were terminated after 1, 4, 24 and 48 h of incubation. For Southern blotting, the cells were washed in PBS, trypsinised, collected and kept at –86°C. For measuring of luciferase activity the cells were solubilised according to manufacturer's instructions (Promega).
Nucleic acid purification from blood and tissue
Total DNA was extracted from blood, tissue and cell samples with a
DNeasy® Tissue Kit (Qiagen) according to the manufacturer's
recommendation. DNA was treated according to the protocol with RNase A
(Qiagen) to remove any RNA. Samples with an A260/A280
ratio between 1.8 and 2.0 were considered pure. DNA concentrations were
measured using NanoDrop® ND-1000. DNA was eluted in nuclease-free water
(Ambion, Austin, TX, USA).
Total RNA was extracted using the Trizol method
(Chomczynski and Sacchi, 1987)
with some modifications. Briefly, tissue samples were homogenised in 1 ml
TRIzol reagent using a rotor-stator homogeniser (UltraThurax; IKA Werke,
Staufen, Germany). For additional removal of DNA and proteins, the water phase
of the initial TRIzol/chloroform separation was added to a second volume of
TRIzol reagent. To remove any contaminating DNA, samples were treated with
DNase (TURBO DNA-free, Ambion) according to the manufacturer's recommendation
and eluted in nuclease-free water. Purified RNA was confirmed to be intact by
gel electrophoresis. Samples with an A260/A280 ratio
between 1.8 and 2.2 were considered pure. RNA concentrations were measured
using the RiboGreen® RNA Quantitation Kit (Molecular Probes, Eugene, OR,
USA) (Jones et al., 1998).
Southern blot analysis
Ten micrograms of total DNA from fish and 2 µg of total DNA from cells
were digested with MluI (Promega) and run on a 0.7% agarose gel. DNA
was blotted to a Nytran N membrane (Schleicher and Schuell, Dassel, Germany)
with a Turboblotter as recommended by the manufacturer and visualised by
enzyme-linked chemiluminescence using digoxigenin (DIG)-labelled PCR probe.
The DIG-labelled PCR probe was constructed using the PCR DIG Probe Synthesis
Kit from Roche (Roche Diagnostics, Mannheim, Germany) and Luc 1 primers
(Table 1), amplifying a 397 bp
fragment in the firefly luciferase gene. Primers were designed using the
Primer Express software (version 2.0; Applied Biosystems) and synthesized by
Operon Biotechnologies Inc. (VWR, West Chester, PA, USA).
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Complementary DNA synthesis
RNA was reversely transcribed according to the manufacturer's instructions
using random hexamers (TaqMan RT-reagents; Applied Biosystems, CA, USA) under
the following conditions; 25°C for 10 min, 37°C for 60 min, 95°C
for 5 min. Reaction volumes were 10 µl and contained 50 ng of total
RNA.
Real-time RT–PCR
Real-time RT–PCRs were performed in duplicate with an ABI PRISM®
7000 Sequence Detection System (Applied Biosystems) at the following cycling
conditions: 50°C for 2 min, 95°C for 15 min, 40 cycles of 95°C for
15 s and 58°C for 60 s. Every PCR contained 2x TaqMan Universal PCR
Master Mix; Applied Biosystems [AmpliTaq Gold DNA Polymerase, dNTPs, passive
reference (ROX) and optimised buffer components], 20x TaqMan Assay Mix,
50 ng of cDNA template and nuclease-free water to a final volume of 25 µl.
Primers and probes were designed and synthesised by Applied Biosystems (Custom
TaqMan® Gene Expression Assays) based on the firefly luciferase gene and
18S rRNA sequence (Acc. No. AF518205; Table
1). The relative expression ratio (R) of the luciferase gene was
calculated based on primer efficiencies (E) and the Ct deviations
(
Ct) of the unknown samples versus a calibrator
after normalisation to 18S rRNA (Pfaffl,
2001). The amplification efficiencies of the primers were assessed
from twofold liver cDNA dilutions. DNA contamination of the RNA was evaluated
by subjecting parallel samples to real-time PCR without preceding reverse
transcription. All data were captured using Sequence Detection Software (SDS
version 1.1; Applied Biosystems).
Extraction and measurement of luciferase protein in cod tissues and in cultures of cod aEEC
Tissue samples from Atlantic cod injected i.m. with pDNA (1.0 mg
kg–1 body mass) were collected 24, 48 and 168 h postinjection
and processed as described previously
(Manthorpe et al., 1993).
Monolayer cultures of aEECs (pooled cells from three fish, approximately
3x105 cells cm2) were established in 9.6
cm2 dishes. The cells were incubated for 1, 4, 24 and 48 h with 10
µg pDNA in 600 µl L-15 medium supplemented with 1% HSA. The experiment
was terminated at different time points by adding 300 µl lysis buffer
(luciferase assay system; Promega, Madison, Wisconsin, USA). Enzyme activity
in the solubilised tissues or cell suspension was measured according to the
manufacturers recommendation (Promega). The results were recorded using a
Luminoscan Ascent® microplate luminometer (Thermo Electron Oy, Vantaa,
Finland). The background, defined as the average luciferase levels plus 2
s.e.m. from tissues or cells that were not injected or incubated with pDNA,
were deducted from all values. The relative light units (RLU) were normalised
to the concentration as determined by the Bio-Rad RC DC protein assay. The
Bio-Rad RC DC protein assay was performed according to the manufacturer's
description (Bio-Rad, Hercules, California, USA). To compensate for the colour
masking/difference of the solubilised tissues known amounts of tissues were
incubated with a fixed amount recombinant luciferase protein (Promega).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
Fluorescent bands were detected only as open circular and supercoiled pDNA
topoforms (data not shown). Fluorescent bands and pDNA disappeared after DNase
treatment. Neves et al. (Neves et al.,
2000) demonstrated a 60% reduction of gene transfer efficiency
when conjugating fluorescent molecules to pDNA by photoactivation. To ensure
that the luciferase gene was capable of expressing the transgene after
labelling pDNA with fluorescein, labelled and unlabelled pDNA was transfected
into semiconfluent Caco-2 cells. The gene transfer efficiency of the labelled
plasmid in Caco-2 cells was reduced by 75% compared to transfection with
non-labelled pDNA.
Anatomical distribution of 125I-fluorescein–pDNA
The anatomical distribution of i.v. and i.m. injected pDNA was investigated
by injecting trap-labelled pDNA. The trap molecule (fluorescein) is unable to
pass through biological membranes and is not actively taken up by cells unless
it is conjugated to a macromolecule. When inside the cell the radioactive trap
label will remain within the cell independent of the fate of the attached
macromolecule. One hour after i.m. injection of trace amounts of
125I-fluorescein–pDNA, approximately 90% of the radioactivity
was recovered from the injection site, 6.6% was found in the blood, 2% in the
heart, 2.3% in the liver and 3.2% in the intestine. Transport of labelled pDNA
(not taken up) from the injection site to other organs studied at 1, 4, 24, 48
and 168 h showed that the labelled pDNA disappeared from the injection site
and accumulated in the intestine, heart and liver almost at the same rate
(Fig. 1). After 48 h
approximately 15% of the radioactivity was found in the intestine, 12% in the
heart and 5.5% in the liver. The specific activity of the heart tissue at 48 h
was much higher than the specific uptake (c.p.m. g–1 tissue)
of the intestine and the liver (1.09x105 c.p.m.
g–1 tissue for the heart tissue compared to
5x103 c.p.m. g–1 tissue for the intestine
and 1.0x 103 c.p.m. g–1 tissue for the
liver). The kidney and spleen contained less than 1% of the recovered label at
all time-points. Total recovered radioactivity was never less than 66% of the
injected dose of labelled ligand.
|
Anatomical distribution of blood-borne pDNA was investigated after i.v. injection of trace amounts of 125I-fluorescein–pDNA, the amount of radioactivity in various tissues was recorded after 5 min, 15 min, 1 h, 4 h, 24 h, 48 h and 168 h. Radiolabelled pDNA was rapidly eliminated from the circulation during the first 15 min of the experiment. The total recovered radioactivity in the blood decreased from 73.2% after 5 min to 14.7% after 60 min and to 2.7% after 168 h (Fig. 2). Approximately 30% of the total radioactivity was found in the heart 5 min after injection of 125I-fluorescein–pDNA; 168 h later the amount increased slightly to 44%. The specific activity in the heart during the same period did not increase. After 15 min the specific activity was 2.66x105 c.p.m. g–1 tissue and 2.71x 105 c.p.m. g–1 tissue after 168 h. The amount of radioactivity found in the kidney and liver was never higher than 3.8% of the total recovered radioactivity for the kidney and 8.6% for the liver. In the spleen and the intestine the amount of radioactivity recovered was low. The high heart to blood ratio (30.3:1) compared with the much lower ratios in liver, anterior kidney and spleen (0.18:1, 1.14:1 and 0.99:1, respectively) indicates a very effective uptake mechanism for pDNA in the cod heart. The total amount of radioactivity found in the carcass after injection of 125I-fluorescein–pDNA decreased from 38% after 5 min to 33% after 168 h. The specific activity in the carcass was never higher than 1.0x102 c.p.m. g–1 tissue.
|
Injection of 125I-fluorescein–pDNA together with excess
amounts of unlabelled pDNA or FSA, a ligand for the cod aEEC scavenger
receptor, effectively blocked uptake of radiotracer by the heart
(Table 2). This indicated that
the uptake of pDNA by the cod heart was specific and was mediated by the cod
aEEC scavenger-like receptor
(Sørensen et al.,
1998). The uptake of radiotracer was inhibited in the kidney as
well, indicating that there was a specific uptake of pDNA in this organ. The
uptake in spleen, liver, intestine and carcass was not influenced by
co-injection of pDNA and FSA. The tissue to blood ratio, after inhibition of
the cod aEEC scavenger-like receptor indicates that the radiotracer, not taken
up by the heart, was not taken up by other organs and most likely continued to
circulate in the blood.
|
Furthermore, we investigated whether 125I-fluorescein–pDNA was bound to or taken up by blood cells or if it remained in the plasma fraction 1 h after i.v. injection. Density gradient centrifugation demonstrated that more than 93% of the radioactivity was present in the serum/plasma fraction and only minor amounts were found in the cellular fractions containing white blood cells (4%) and red blood cells (0.5%).
In vivo and in vitro stability of native pDNA in cod blood
The degradation of pDNA in blood in vivo and in vitro was
investigated. The supercoiled topoform of pDNA was completely changed to open
circular and linear topoforms within 5 min after injection and then degraded
to low molecular mass products (Fig.
3A). The in vitro degradation of pDNA by blood was
similar (Fig. 3B).
|
Southern blot analysis of cod tissues and cells
The topoform of the re-isolated pDNA in blood, liver, kidney, spleen,
heart, and injection site was examined after injection of pDNA (1 mg
kg–1 body mass). Southern blot revealed extra-chromosomally
super-coiled, open circular and linear pDNA in all the investigated tissues 48
h after i.v. injection. Degradation products were only detected in the muscle
tissue at the site of injection (Fig.
4). No pDNA was detected in tissues from PBS injected fish.
|
Enzymatically active luciferase was observed in muscle tissue at the injection site 168 h after injection. In the muscle tissue we measured 115.71±65 relative light units (RLU) per mg protein, compared to 0.25 RLU mg–1 protein in the control fish. In heart, kidney, spleen, liver and muscle tissue no expression of luciferase protein was found.
Cellular distribution of rhodamine–pDNA
Tissue sections prepared from fish 1 h after i.v. injection with
fluorescein–pDNA revealed the presence of fluorescence in endocardial
cells in both the atrium (Fig.
5A) and the ventricle (Fig.
5B).
|
Endocytosis of rhodamine–pDNA in cod aEEC and head kidney leukocytes in vitro
Cultures of aEECs (Fig. 6A)
and head kidney leukocytes were incubated with rhodamine–pDNA for 2 h.
Subsequent fixation and examination in the fluorescence microscope revealed
that all cells accumulated large amounts of fluorescent Rhodamine in distinct
vesicles.
|
|
|
Southern blot analysis of endocytosed pDNA in cod aEEC in vitro
Southern blot analyses were performed on solubilised cells 1, 4, 24 and 48
h after incubation of 10 µg pDNA. Southern blot analysis of cultured aEEC
showed a complete degradation of supercoiled, open-circular and linear pDNA to
low molecular mass products after 24 h
(Fig. 9). No pDNA was detected
in control aEEC or in the last PBS washing solution, prior to DNA
isolation.
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The present results revealed that trace amounts of
125I-fluorescein–pDNA after i.m. injection disappeared from
the injection site and accumulated almost at the same rate in the heart, liver
and intestine. The use of a radiolabelled fluorescein adduct that is trapped
after endocytosis (Li et al.,
2003), with almost no escape of the label from the intracellular
compartments, allows a reliable measurement of the anatomical and cellular
site of uptake of pDNA. Local degradation of pDNA in the muscle tissue (at the
site of injection) of mice has previously been reported by
(Barry et al., 1999;
Manthorpe et al., 1993). In
those studies, 95–99% of the injected pDNA was degraded and was no
longer present in tissue 90 min after injection, as detected by Southern blot
and PCR. In our experiment, Southern blot analysis of muscle tissue from the
injection site showed presence of degradation products in addition to intact
pDNA 48 h after injection. A total of 30% the radiolabel escaped from the site
of injection during a period of 48 h. Furthermore, the finding of 10% of the
recovered radioactivity in the cod heart after 48 h indicated that a part of
the injected pDNA escaped the local degradation and entered the blood where it
was rapidly cleared by the cod heart cells. This result was confirmed by
Southern blot analysis that demonstrated intact pDNA in heart tissue 48 h
after injection. The 125I-fluorescein has been shown not to be
recognised or taken up by the heart
(Sørensen et al.,
1997a). The radioactivity in the intestine and liver after i.m.
injection may be a result of drainage of low molecular mass
125I-fluorescein or 125I-fluorescein-labelled
oligonucleotides from the injection site to the general circulation where it
was secreted by the kidney or bile to the intestine. However, Southern blot
analysis of liver and intestine tissues showed the presence of intact pDNA.
Accumulation of macromolecules in the intestine after intravenous injection
has been reported previously by Dalmo et al.
(Dalmo et al., 1996).
Gradient centrifugation of the blood after injection of
125I-fluorescein-pDNA showed that 90% of the radioactivity was
present in the plasma fraction. An in vitro incubation of pDNA with
whole blood or plasma showed a complete degradation of the pDNA within
minutes. These findings, together with the observation that the amount of
radiolabel in the blood was low at all time-points investigated, suggested
that the transit time of labelled pDNA or its degradation products in the
circulation was very short. Sørensen et al.
(Sørensen et al.,
1997a) reported that subcutaneously injected hyaluronan (a
connective tissue macromolecule), was transported to the blood circulation
before being finally eliminated by cells in the cod heart. In their experiment
65% of the injected radiolabelled hyaluronan was found in the heart after 48
h.
Intravenously administered 125I-fluorescein–pDNA was
rapidly eliminated from the circulation and mainly taken up by the cod heart,
where approximately 25% of the total recovered radioactivity was found 5 min
after administration. The amount of radioactivity recovered from the heart at
later time-points was essentially the same. This result confirmed the high
blood clearance capacity and the efficiency of the cod aEEC system. The
maximum clearance capacity of these cells was not tested in this study but the
total amount recovered from the heart after injecting 10 000 c.p.m.
µg–1 pDNA in the ligand competition study corresponded to
a total uptake of 25 µg DNA compared to approximately 0.25 µg in the
initial study. This indicated that most of the injected pDNA was cleared from
the circulation within 5 min. The amounts of radioactivity found in kidney,
spleen, liver and intestine were lower compared with the amounts found in the
heart. The total amount of radioactivity found in the carcass was high, but
the specific activity was less than 7.1x103 c.p.m.
g–1 tissue compared with 3217x103 c.p.m.
g–1 tissue in the heart tissue. The efficient uptake of
macromolecules by the cod heart has been reported earlier. Intravenously
injected radiolabelled gelatinised cod skin collagen was rapidly eliminated
from the blood (t
=15 min), and taken up by the
heart, with minor uptake in other organs
(Smedsrød et al.,
1995). The cod heart is the most important scavenger organ for
circulating FSA, aminoterminal propeptides of type 1 procollagen (P1NP) and
bacterial lipopolysaccharide (LPS) all of which are ligands for the scavenger
receptors. In salmonid fish, the kidney tissue has been reported to be the
major organ for DNA clearance, followed by the liver, blood, muscle and
gonadal tissue after i.v. administration
(Nielsen et al., 2006). In
mammals, the liver is the main organ responsible for the rapid clearance of
DNA from the circulation. The non parenchymal cells of these mammals are
mainly responsible for this clearance, with the liver sinusoidal endothelial
cells as the major contributor (Hisazumi
et al., 2004). Uptake of DNA in other organs such as kidney and
bone marrow has also been reported
(Bijsterbosch et al.,
1997).
Fluorescence microscopy showed that rhodamine pDNA was taken up by
endocardial endothelial cells lining the muscular trabecula of both heart
chambers. The endocytic (high blood clearance) capacity of the endocardial
endothelial cell of cod has been well studied. These special endocardial
endothelial cells, together with the sinusoidal endothelial cells of the
mammalian liver and salmonid kidney have been characterized as scavenger
endothelial cells (SEC). Using specific and extremely effective endocytosis
they eliminate soluble waste macromolecules from the circulation at a very
high rate (Seternes et al.,
2002). In vitro studies in cultured atrial endocardial
cells have provided functional evidence for the existence of distinct
scavenger, mannose and hyaluronan receptors in these endocytically highly
active cells (Seternes et al.,
2001b; Sørensen et al.,
2001; Sørensen et al.,
1998). When trace amounts of radiolabelled DNA was co-injected
with excess amounts of unlabelled pDNA or FSA, the tissue to blood ratio
measured after 1 h in the atrium and ventricle was reduced by more than 90%,
indicating that the uptake of pDNA in the cod heart was specific and largely
mediated via a functional scavenger receptor. The observation that
the in vitro uptake of 125I-fluorescein pDNA was
effectively inhibited by excess amounts of scavenger receptor ligands supports
the idea that pDNA was taken up via the functional scavenger receptor
expressed by these cells. Sørensen et al.
(Sørensen et al., 1998)
and Seternes et al. (Seternes et al.,
2001a) have demonstrated the presence of a functional scavenger
receptor in cultures of aEEC by ligand competition studies. Similar studies in
cultures of rat liver sinusoidal endothelial cells (sLEC) have suggested that
the scavenger receptor was responsible for cellular binding and uptake of
naked DNA in both rats and mice (Hisazumi
et al., 2004).
Ligands taken up by the cod aEEC are subjected to intracellular degradation
in endosomes and lysosomes. Southern blot analysis of solubilised cod aEEC
cells after incubation with unlabelled pDNA showed total degradation of pDNA.
Earlier studies by Sørensen et al.
(Sørensen et al., 1998)
showed that as much as 45% of the added 125I-FSA and
125I-PINP were degraded and recovered as low molecular mass
products after 24 h of incubation with cultured aEEC. Moreover, hyaluronan, a
connective tissue polysaccharide, was taken up by the aEEC in vitro
and degraded to acetate (Seternes et al.,
2001b). Another ligand for the aEEC scavenger receptor,
lipopolysaccharide was not degraded by the aEEC even after 48 h incubation
(Seternes et al., 2001a). All
together, the aEEC shows an efficient endocytic and degradative capacity
towards a whole range of macromolecules. Degradation of pDNA have been
demonstrated in primary cultures of rat sinusoidal endothelial cells by
release of acid-soluble radioactivity from the cells after incubation with
32P-pDNA (Hisazumi et al.,
2004). The use of the trap-labelled fluorescein in our study
prevented us from measuring acid-soluble radioactivity in the culture media.
Expression of the luciferase reporter gene was investigated in cod aEEC after
incubation with unlabelled pDNA by real-time RT–PCR and luciferase
enzyme assay. The construct used in this study was shown to give expression of
luciferase protein in cod muscle tissue 168 h after i.m. injection. Our
results showed that neither luciferase mRNA nor enzyme were present in the
cultured aEEC, even after 48 h of incubation. These results were in accordance
with the i.v. and i.m. injection results that demonstrated high amounts of
pDNA in the heart but no expression of mRNA and luciferase protein.
The total recovered radioactivity in the kidney was low, but there was
still a reduction after co-injection of unlabelled pDNA and FSA. The anterior
kidney of Atlantic cod and other teleosts is a haematopoietic organ and a rich
source of macrophages (Braun-Nesje et al.,
1981; Sørensen et al.,
1997b). These results indicated that the cod macrophage was
involved in the clearance of circulatory pDNA. A functional scavenger receptor
responsible for uptake of particles in fish macrophages was reported in
rainbow trout macrophages by Frøystad et al.
(Frøystad et al.,
1998). However, this macrophage receptor seemed to be of minor
importance in the blood clearance of soluble scavenger receptor ligands
(Seternes et al., 2001a;
Sørensen et al., 1998).
In vitro studies of head kidney leukocytes from cod demonstrated that
these cells have the capability to bind and endocytose pDNA. In spite of these
results, that the head kidney cells can take up pDNA from the circulation, the
results from the distribution studies with
125I-fluorescein–pDNA strongly suggest that the head kidney
leukocytes contributed less to the total in vivo blood clearance of
pDNA in cod. The uptake of pDNA in cultures of aEEC is rapid, with
approximately 33% of the total added 125I-fluorescein–pDNA
taken up after 48 h. However, compared with the uptake rate of other SR
ligands (added in the same concentration, approximately 1x106
c.p.m. µg–1 protein) by the aEEC it is markedly slower.
Already 1 h after incubation of 125I-FSA the uptake reached 25%,
increasing to 75% after 6 h
(Sørensen et al.,
1998). The reason for the difference in rate of uptake was not
investigated. The uptake of 125I-fluorescein–pDNA by the head
kidney leukocytes was similar with approximately 29% of the total added
radioactivity taken up after 48 h.
Based on the results from the present and earlier studies, it seemed that there was drainage of intact pDNA from the site of injection to the general blood circulation. The transit time of pDNA in the circulation was very short, and even if the pDNA was subjected to degradation, most of the blood-borne pDNA was cleared by specific uptake by scavenger endothelial cells lining the muscular trabecula of both heart chambers. The uptake of pDNA was inhibited both in vivo and in vitro by co-injecting ligands recognised by scavenger receptors. The endocytosed pDNA was degraded by the aEEC in vitro. There was no expression of the luciferase mRNA or protein in the cod aEEC. The cod head kidney macrophage was able to take up pDNA by a specific mechanism in vitro. However, the head kidney macrophage did not seem to be involved in the in vivo clearance of pDNA from the blood.
|
Acknowledgments |
|---|
|
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