V. MATERIALS & METHODS
V.1 MATERIAL
V.1.1. Chemicals and reagents
All chemicals and reagents were purchased from Merck (Darmstadt), Roche (Mannheim), Roth (Karlsruhe), Serva (Heidelberg), and Sigma (Steinheim) if not otherwise mentioned.
Enzymes
Alkaline phosphatase Biolabs, Frankfurt am Main Kollagenase Biochrome, Berlin
Proteinase K (PCR grade) Roche Diagnostics, Mannheim Restriction endonucleases Biolabs, Frankfurt am Main Taq-Polymerase Biolabs, Frankfurt am Main T4 DNA ligase Roche, Mannheim
Kits
ECL Western Blotting Reagent Pierce, USA
Qiaquick Gel Extraction Kit Qiagen, Hilden Plasmid Mini/Midi/Maxi Kit Qiagen, Hilden Readyprime II Random Prime Labeling System Amersham, Heidelberg
Other Chemical substances and inhibitors
Table 1. The working concentrations were determined either by dose escalation studies or adapted from the literature, all substance were stored at -20°C except for CTB-FITC (4°C).
Name Working concentration
Dissolved in
Manufacturer
Brefeldin A (BFA) 10 µg/ml DMSO Sigma, Deisenhofen Cholera toxin
subunit B (CTB)-FITC
5 µg/ml H2O Sigma, Deisenhofen
Cytochalasin D 20 µM DMSO Sigma, Deisenhofen
Fumonisin B1 50 µM DMSO Sigma, Deisenhofen
Fluorescein-diacetate
ca. 1 µg/ml DMSO Sigma, Deisenhofen
Methyl-ß-cyclodextrin
10 mM H2O Sigma, Deisenhofen
Mevinolin 10 µM DMSO Sigma, Deisenhofen
Myriocin 20 µM DMSO Biomol, Hamburg
Nocodazole 20 µM DMSO Sigma, Deisenhofen
Paclitaxel 20 µM DMSO Sigma, Deisenhofen
Phalloidin-FITC/TRITC
4 µg/ml H2O Molecular Probes,
UK
V.1.2. Bacterial strains
Top 10 F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacΧ74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL (StrR) endA1 nupG (Invitrogen, Karlsruhe)
DH5α F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 tonA (Gibco BRL, Eggenstein)
V.1.3. Cell culture
Cell lines and primary cells
LMH Chicken hepatoma cell line
D2 LMH cells stably transfected with DHBV-genome, kindly provided by J. Summers and W. Mason, USA
PDHs Primary duck hepatocytes isolated from Pekin duck foetuses
Liver biopsies from adult Pekin ducks
Reagents for cell culture
Cells were cultured at 37°C with 5% CO2.
All cell culture reagents were purchased from Gibco BRL (USA), Sigma (Mannheim), or Biochrom (Berlin). The media were supplemented as follows:
Dulbecoo’s Modified Eagle’s Medium (DMEM) for LMH and D2 cells
10% Heat inactivated FCS 100 U/ml Penicillin
100 µg/ml Streptomycin
1mM Sodium pyruvat
1% Non essential amino acids
Williams’ medium E for PDHs
2 mM L-glutamine
15 mM HEPES, pH 7.2
1.5% DMSO
10 µM Hydrocortisone
1 nM Insulin
100 U/ml Penicillin 100 µg/ml Streptomycin
For temperature block experiments, cells were cultured in a CO2-independent medium (Invitrogen, Karlsruhe) supplemented as described for DMEM medium for D2 cells or for Williams’ medium E for PDHs.
Trypsin solution
0.25% Trypsin
1 mM EDTA
Tissue culture lab ware
All cell culture plastic ware was purchased from Greiner (Solingen), and Sarstedt (Nuembrecht).
V.1.4. Antibodies
Primary antibodies
Name Species Dilution
for WB/IF
origin
anti-actin mouse WB 1:10000 Sigma, Deisenhofen anti-albumin rabbit WB 1:5000 Nordic immunology,
Offenbach anti-α-tubulin mouse WB 1:10000
IF 1:1000
Sigma, Deisenhofen
anti-apolipo- protein A-I
rabbit WB 1:5000 M. Hermann, Vienna
anti-calnexin rabbit WB 1:5000 IF 1:200
Stressgen, Canada
anti-core rabbit WB 1:10000 IF 1:400
L. Cova, Lyon, France
anti-EEA1 rabbit WB 1:2500 IF 1:200
Stressgen, Canada
anti-gamma- adaptin
rabbit WB 1:2000 IF 1:200
R. Prange, Mainz
anti-GFP rabbit WB 1:5000 Santa-Cruz, California anti-L 1H1 mouse IF 1:100 (182)
anti-L KpnI rabbit WB 1:10000 IF 1:800
(183)
anti-membrin mouse WB 1:5000 IF 1:200
Stressgen, Canada
anti-MTP rabbit WB 1:5000 IF 1:200
M. Hermann, Vienna
anti-PDI mouse WB 1:5000
IF 1:200
Abcam, UK
anti-rab5B rabbit WB 1:5000 IF 1:200
Santa-Cruz, California
anti-duck S rabbit WB 1:2500 IF 1:100
H. Schaller, Heidelberg
Table 2. Primary antibodies diluted in TBST containing 3% blocking milk. Abbreviations WB: western blot; IF: immunofluorescence.
Secondary antibodies
Name Species Dilution Manufacturer
anti-rabbit-HRPO goat WB 1:10000 Dianova, Hamburg anti-mouse-HRPO goat WB 1:10000 Dianova, Hamburg anti-goat-HRPO doncky WB 1:5000 Dianova, Hamburg anti-rabbit-488
(Alexa 488)
goat IF 1:800 Molecular Probes, Netherlands anti-rabbit-594
(Alexa 594)
goat IF 1:800 Molecular Probes, Netherlands
anti-rabbit-555 (Alexa 555)
goat IF 1:800 Molecular Probes, Netherlands
anti-mouse-488 (Alexa 488)
goat IF 1:800 Molecular Probes, Netherlands
anti-mouse-594 (Alexa 594)
goat IF 1:800 Molecular Probes, Netherlands anti-mouse-555
(Alexa 555)
goat IF 1:800 Molecular Probes, Netherlands
Table 3. Secondary antibodies
The secondary antibodies coupled with HRPO were dissolved in sterile 50% glycerol according to the manufacturer instructions and diluted in TBST containing 3% blocking milk.
Antibodies coupled with fluorophores were diluted in 1 x PBS. Abbreviations WB: western blot; IF:
immunofluorescence
V.1.5. Primers
Oligonucleotides were purchased from Invitrogen (Karlsruhe) or MWG (Ebersberg).
Analytical primers
Both primers were adapted from (66).
DHBV P1 5’-GCG CTT TCC AAG ATA CTG GAG CCC AA-3’
DHBV P2 5’-CTG GAT GGG CCG TCA GCA GGA TTA TA-3’
Primers for cloning
Construct Primer name Restriction site Sequence 5’-3’
EGFP-S Forward EGFP-S BglII GCG AGA TCT ATG TCT GGT ACC TTC GGG GGA
Reverse EGFP-S SacII GCG CCG CGG CTA ACT CTT GTA AAA AAG AGC
AGA
S-EGFP Forward S-EGFP EcoRI GCG GAA TTC ATG TCT GGT ACC TTC GGG GGA
Reverse S-EGFP SacII GCG CCG CGG ACT CTT GTA AAA AAG AGC
V.1.6. Plasmids
Commercially available plasmids
pcDNA 3.1 (-) Invitrogen, Karlsruhe
pEGFP-N1 BD Biosciences/Clontech, USA
pEGFP-C1 BD Biosciences/Clontech, USA
YFP-ß-galactosidase BD Biosciences/Clontech, USA
Provided plasmids
RFP-CD63, RFP-CD82, YFP-Rab11, YFP-Rab7, and Tsg101-YFP were generously provided by W. Mothes, New Haven, USA (157).
VSV-G-GFP: encodes the glycoprotein G of the vesicular stomatitis virus (VSV) within the pEGFP-N1 vector (H. Sirma, HPI, Hamburg).
DHBV wt: encodes a 1.2-fold genome of DHBV, pDHBV16/1.1 (184).
DHBV 16t-27: encodes the DHBV 16 genome in tandem (185).
Generated plasmids
Name Vector Insert Resistance Origin/Reference
EGFP-S pEGFP-C1 Duck S sequence
amplified by PCR, cloned via BglII and SacII
Kanamycin L. Lin. and H. Sirma HPI, Hamburg
pDuck-S pcDNA 3.1 Duck S (generated by the transfer of the S coding region from SEGFP into pcDNA3 via BamHI and ApaI)
Ampicillin M. Mhamdi, HPI, Hamburg
pGEM-D1OG
(Expression vector for duck preS/S)
pGEM13 zf (+)
D10G (DHBV full length monomere), cloned via EcoRI
Ampicillin N. Lohrengel, HPI, Hamburg
SEGFP pEGFP-N1 Duck S sequence
amplified by PCR, cloned via EcoRI and SacII
Kanamycin M. Mhamdi, HPI, Hamburg
V.1.7. Devices
Centrifuges
Mini ultrastrifuge, Sorvall Discovery M120 Hitachi, Japan
RC-5B refrigerated super speed centrifuge Sorvall, Bad Homburg with the rotors GSA, SS-34
Table Centrifuge 5415 R (refrigerated) Eppendorf, Hamburg Table Centrifuge 5415 C Eppendorf, Hamburg Ultracentrifuge Optima LE-80K Beckman, USA with the SW-41 rotor
Centrifugation tubes for SW 41, 14 ml Beckman, USA
Microscopes
Light microscope Leica, Bensheim Fluorescence microscope Zeiss, Jena CLSM LSM 510 Zeiss, Jena
Other devices
Film developer Curix 60 Agfa, PMA Bode, Hamburg Fluor-S multi-imager Bio-Rad, USA Fraction recovery system Beckman, USA
Gel documentation system Decon, Hohengandern Homogenisator GlasCol, USA
Image-sacnner Epson, Meerbuch Incubator (for cells) Thermo, Berlin
Incubator shaker New Brunswick scientific, USA pH meter Inolab, Germany
Phosphoimager Fujix Bas Raytest, Straubenhardt Photometer Ultraspec 3000 pro Pharmacia, Freiburg
RoboCycler Gradient 40/96 Stratagene, Netherland Semi-Dry-System Trans blot SD Bio-Rad, USA
SDS-PAGE device Bio-Rad, USA
Shaker IKA, Staufen
Sterile bench Thermo, Berlin Thermomixer compact Eppendorf, Hamburg UV-Stratalinker Stratagene, Netherland Water bath Milian, France