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MATERIALS AND METHODS
Mice and immunization. All animals used in this study were obtained from
the Jackson Laboratory and were housed at the central animal care facility at the
University of Rostock. All procedures and assays were approved by the competent
state's Animal Care Committee and followed the guidelines for the care and use of
laboratory animals.
CIA was induced in animals according to established protocols previously
described by our group (6). In brief, (DBA/1J×FVB/N) F2 progeny were immunized at
8-12 weeks of age at the base of the tail with 125 µg of bovine collagen II (Chondrex)
dissolved in 50 µL of 0.1 M acetic acid and mixed with an equal volume (50 µL) of
CFA (IFA with 4 mg/mL Mycobacterium tuberculi; DIFCO Laboratories Detroit, USA).
Three weeks after immunization and before clinical signs of arthritis manifested, in
vivo multifluorescence microscopy was performed.
In vivo multifluorescence microscopy. Animals were anaesthetized with
ketamine (90 mg/kg body weight) and xylacin (6 mg/kg) and placed on a heating pad
to maintain their body temperature at 37 °C. A catheter was placed in the left jugular
vein for application of fluorescent dyes. For in vivo multifluorescence microscopy of
synovial microcirculation, we used the knee joint model initially described by
Veihelmann and coworkers (8) and adopted by our group (9). Briefly, the skin was
incised distal to the patella tendon. After removal of the overlying soft tissues, the
patella tendon was transversally cut and the proximal and distal part carefully
mobilized. After exposure, the 'Hoffa's fatty body' was superfused with 37 °C warm
physiological saline solution to prevent the tissues from drying and finally covered
with a glass slide. Following a 15 min stabilization period after surgical preparation, in
vivo microscopy of the synovial tissue was performed. At the end of the experiments,
animals were killed by exsanguination.
Microcirculatory analysis. For quantitative offline analysis a computerassisted microcirculation image analysis system was used (CapImage v7.4; Zeintl,
Heidelberg, Germany). Functional capillary density (FCD) was defined as the total
length of red blood cell (RBC)-perfused capillaries per observation area, and is given
in cm/cm2. For assessment of leukocyte-endothelial cell interaction in postcapillary
venules, flow behaviour of leukocytes was analyzed with respect to free floating,
rolling and adherent leukocytes. Rolling leukocytes were defined as those cells
moving along the vessel wall at a velocity less than 40 % of the leukocytes at the
centre line, and are expressed as a percentage of the total leukocyte flux. Venular
leukocyte adherence was defined as the number of leukocytes not moving or
detaching from the endothelial lining of the venular vessel wall during an observation
period of 20 s. Assuming cylindrical microvessel geometry, leukocyte adherence was
expressed as non moving cells per endothelial surface (n/mm 2), calculated from the
diameter and length of the vessel segment analysed. In postcapillary venules, centre
line RBC-velocity (VRBC) was determined using the line shift method (CapImage;
Zeintl, Heidelberg, Germany).
Gene selection and quantitative PCR. Based on our previously published
and unpublished experimental data (9), we selected eleven genes (Cd44, Il13r1,
Ccr3, Defb3, Sele, Sell, Selp, Xcl1, Il1, Tnf and Ifn) which are differently
expressed at the early stage of arthritis and/or are known to be involved in cell
adhesion. Paws and lymph nodes were removed after IVM. Snap frozen paws were
homogenized using mortar and pestle and lymph nodes were homogenized using
FastPrep instruments. Total RNA was extracted with the RNeasy Mini Kit (Qiagen,
Germany) according to the manufacturer’s instructions. For reverse transcription, we
used 300 U of SUPERSCRIPTTM RNase H- Reverse Transcriptase, 20 U of RNasin,
3 µM random hexamers (Amersham Pharmacia Biotech), deoxynucleoside
triphosphate, dithiothreitol and 2 µg of RNA sample per 25 µL reaction volume.
Gene quantification was performed on the ABI Prism 7700 Sequence
Detection System (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany).
TaqMan primers and probes were purchased from Perkin-Elmer Applied Biosystems.
Quantitative PCR was performed with 50 ng of cDNA according to the manufacturer’s
instructions in a final volume of 12.5 µL. Thermal cycling conditions were as follows:
2 min at 50 °C, 10 min at 95 °C followed by 45 - 50 repeats of 15 s at 95 °C, and 1
min at 60 °C. In each run a negative control (distilled water) was included. For each
RNA isolation measurements of gene expression were taken two times, and the
means of these values were used for further analysis.
According to the manufacturer’s User Bulletin #2 (Applied Biosystems) the
comparative Ct method and the internal control (GAPDH) were used to normalize the
expression levels of target genes.
Genomic screening. For the genetic analysis 132 informative microsatellite
markers covering the genome to the extent of 94% with average inter-marker
distance of 11.22 cM were used. The accuracy of our loci order and interval maps
was verified by comparing the genetic map calculated by our data with the Mouse
Genome Informatics map. Microsatellite markers were chosen from Jackson mouse
database (www.jax.org) and were tested if they are informative for the DBA/J1 and
FVB/N strains. A complete list of markers is available upon request. Primers for
informative microsatellite markers were ordered from Sigma-Genosys (Steinheim,
Germany). The genomic DNA used for genotyping the mice was isolated from a 1 cm
tail tip by using standard isolation protocols (17). Genomic DNA from each animal
was genotyped for 132 microsatellite loci by using PCR amplification: Genomic DNA
(20 ng) was amplified in a final volume of 10 µL containing Hot Start Taq polymerase
(0.25U) (Qiagen), primers (0.1 µM each), 50 mM KCl, 10 mM Tris, 2.5 mM MgCl2, 0.2
mM dNTP, and 0.02 µM M13-IRD700 or M13-IRD2 or M13-IRD4. Amplification
conditions were as follow: 95 °C for 15 min, followed by 2 cycles of 94 °C for 30 s, 57
°C for 1 min, 72 °C for 1 min, then another 37 cycles of 94 °C for 30 s, 55 °C for 1
min, 72 °C for 1 min, and a final extension at 72 °C for 7 min. The reactions were
performed using GeneAmp PCR System 9700 cycler (Applied Biosystems, Inc.,
Foster City, CA). The PCR products were analysed with a capillary electrophoresis
sequencer (CEQ 8800; Beckman Coulter) according to the manufacturer's
instructions. The genotypes were scored using the program FRAGMENT ANALYSIS
supplied by Beckman Coulter.
Linkage Analysis. All linkage analyses have been made with QTX Map
Manager software. The order of the loci was obtained from the Mouse Genome
Informatics map. Rolling and adherent leukocytes, functional capillary density, V RBC,
capillary width and the gene expression level data were taken as phenotypes.
Continuous trait values were checked for normal distribution and logarithmic values
were used when it was necessary. Outliers were detected using Grubb´s test at the
95 % significance level.
As the significant and suggestive linkage threshold values, we have followed
the guidelines from the permutation test of data (n = 1000). The association between
markers and phenotypes were also tested by F statistic (ANOVA analysis)
(KaleidaGraph software).