at 4 C for at least 30 min. If the concentration of viruses in the tested solution is high enough, the erythrocides are agglutinated which results in a diffuse red staining, otherwise the erythrocides sink to the bottom of the well. The haemagglutination titer denotes the reciprocal value of the dilution at which hemagglutination can still be observed.
3.2.4 Viral titration by plaque-forming assay
To determine the amount of infectious virus particles in solutions as cell supernatants or allantoic fluids, a plaque-forming assay was performed. When infecting a confluent cell monolayer with influenza virus, the cytopathic lytic effect of the virus on infected cells results in the formation of visible plaques that can be counted, to determine viral titers.
One day prior to virus titration, MDCK cells were seeded in 12-well plates, so that they were confluent on the day of titration. MDCK cells were then infected with serial dilutions of virus containing solutions as described in section 3.2.1 and overlaid with Avicel medium containing 1 g=ml TPCK trypsin for 48 h at 37 C, 5 % CO2. Avicel medium was removed and cells were washed twice with PBS following fixation and staining with 0.1 % crystal violet in 10 % formaldehyde for at least 15 min at RT. Staining solution was removed by water washes and cells were dried at RT. Viral titers were determined by multiplying the number of plaques per dilution with the reciprocal dilution factor times 10 (for infection with 100 l virus dilution) and are displayed as PFU=ml.
3.3 Molecular biology methods
3.3.1 Polymerase chain reaction
The polymerase chain reaction (PCR) is a technique for amplification of specific DNA-segments by using two short oligonucleotides (primers) that are complimentary to the 3’ ends of the sense and antisense strand of the target gene. The techincal process is as follows: In a first step, the DNA template is denatured by heat to facilitate binding of the primers to the DNA strands. Then, beginning from the hybridized primer sequences, a special thermostable DNA polymerase synthesizes new DNA strands complementary to the DNA template by adding dNTPs in 5’ to 3’ direction. These three steps, denaturation, annealing and elongation, are repeated for a defined number of cycles to amplify the target DNA sequence exponentially.
To generate the pEGFP-C1-PKR plasmid for the expression of GFP-tagged PKR, the human PKR-gene was amplified from pcDNA3.1-V5-PKR template and BamHI and XhoI restriction sites were added in a PCR reaction using the Expand™ High Fidelity PCR System (Roche) according to the manufacturer’s instructions. The following reaction parameters were em-ployed:
5 x reaction buffer with 15 mMMgCl2 10 l Template DNA pcDNA3.1-V5-PKR (c=50 ng= l) 1 l
Primer 1 XhoI-PKR-For (c=10 M) 2 l
Primer 2 BamHi-PKR-Rev (c =10 M) 2 l
Nucleotide Mix (c =10 mM) 0.5 l
Expand High Fidelity Enzyme Mix 0.5 l
ddH2O ad 50 l
PCR programme:
Number of cycles Temperature Time Step
1x 95 C 30 s Initial denaturation
95 C 30 s Denaturation
35x 55 C 30 s Primer annealing
72 C 4 min Elongation
1x 72 C 10 min Final elongation
1x 4 C 1 Cooling
3.3.2 PCR purification and DpnI digestion
PCR products were purified from excessive primers, nucleotides and enzymes after PCR or restriction enzyme digestion using the QIAquick®PCR Purification Kit according to the manufacturer’s instructions. Purified DNA was eluted with 30 l to 50 l double destilled (dd)H2O.
To degrade remaining template DNA, a DpnI digestion was performed following the PCR reaction. DpnI is a restriction enzyme that specifically degrades methylated DNA. The parental template DNA is from bacterial origin and is therefore methylated. In contrast to that, the newly PCR-generated DNA does not have any methylation modification and will not be degraded in the DpnI reaction. To perform the restriction analysis, 2 l DpnI, 6 l 10x Tango buffer and 2 l were added to 50 l PCR reaction and incubated for 2 h at 37 C.
3.3.3 Agarose gel electrophoresis
DNA and RNA segments can be separated according to their size and form by agarose gel electrophoresis. To generate 1 % agarose gels, 1 g agarose was boiled in 100 ml 1x TBE buffer, cooled down and 6 l of Midori green were added to stain DNA. Samples were supplemented with 6x loading buffer. Separation of samples was performed at a constant voltage of 100 V and DNA was visualized on a transilluminator with UV light. Size and concentration of samples was determined by comparison to commercially available DNA ladders.
42 3.3. Molecular biology methods
3.3.4 Restriction enzyme digestion
Retriction endonucleases are bacterial enzymes that cut the phosphodiester bonds of the dsDNA backbone at specific DNA sequences, referred to as “enzyme recognition sequences”.
By separating the phosphodiester bonds between two nucleotides, the enzymes generate blunt ends or 3’-overhangs, creating the possibility of ligating two separate DNA segments with complementary overhangs.
In this thesis, restriction enzyme digestion of PCR products or plasmid vectors was perfomed according to the manufacturer’s protocol in a total volume of 20 l with 2 l restriction enzyme and 2 l of the corresponding 10x digestion buffer at 37 C.
3.3.5 Vector dephosphorylation
To further prepare the plasmid vector for ligation with the PCR-generated DNA fragment, 5’-phosphate groups were removed by applying calf-intestinal alkaline phosphatase (CIAP) to prevent religation of the linearised vector DNA and to increase the yield of insert containing expression vector.
Dephosphorylation reaction was performed according to manufacturer’s instructions in a 50 l reaction volume with 5 l CIAP enzyme (c =20U= l) and 5 l 10x reaction buffer for 30 min at 37 C.
3.3.6 Ligation
DNA fragments with complimentary nucleotide overhangs, created by restriction enzyme digestion (see section 3.3.4), can be ligated by employing a DNA ligase. In this thesis, purified XhoI and BamHI digested PKR-DNA was ligated with equally treated pEGFP-C1 plasmid vector at a ratio of 1:1. Taking into account the different sizes of plasmid vector and DNA insert, 50 ng of pEGFP-C1 vector were ligated with 18 ng insert. Ligation was performed ON at 16 C by following protocol:
DNA insert 18 ng
plasmid vector 50 ng T4 DNA ligase 2 l 10x ligase buffer 1 l
ddH2O ad 10 l
3.3.7 Transformation of competent bacteria
Transformation is the process of taking up circular plasmid DNA in competent bacterial cells. To facilitate selective growth of transformed bacteria, the inserted plasmids contain antibiotic resistence genes, so that only bacteria carrying the plasmid DNA can grow in media supplemented with the corresponding antibiotic.
For one transformation reaction, 50 l competentE. coli XL-1 BlueorBL26cells were thawed on ice, mixed with 5 L of ligation reaction (see section 3.3.6) or 0.5 L expression plasmid and inubated on ice for 30 min. Then, heat shock for the induction of DNA uptake was performed for 90 s at 42 C, cells were incubated on ice for 2 min and 500 l super optimal broth with catabolite repression (SOC) medium was added, followed by incubation at 37 C for 30 min with gentle shaking. Bacteria were plated on 2xTY agar plates supplemented with antibiotic (100 g=ml Ampicillin or 50 g=ml Kanamycin) and incubated ON at 37 C.
3.3.8 Plasmid preparation
To isolate plasmid DNA, single colonies of transformed bacteria were picked and grown ON at 37 C, 200 rpm in 5 ml (“mini culture”) or 200 ml (“maxi culture”) 2x TY medium contain-ing appropriate antibiotic (100 g=ml Ampicillin or 50 g=ml Kanamycin). Bacteria were pelleted and plasmid DNA was isolated employing the Invisorb®Spin Plasmid Mini Two Kit (Stratec) for mini cultures or the QIAfilter Plasmid Maxi Kit for maxi cultures according to the manufacturer’s instructions. DNA was eluted with 50 L ddH2O (mini culture) or 200 L ddH2O (maxi culture) and DNA concentration was determined using the NanoDrop 8000 machine (Thermo Fisher Scientific).
3.3.9 DNA sequencing
DNA was sequenced by Sanger sequencing using the BigDye® Terminator 3.1 Cycle Se-quencing Kit (Applied Biosystems) according to the manufacturer’s instructions. Following protocol and PCR programme were used:
5 x ABI reaction buffer 1.5 l
Template DNA 200 ng
Sequencing primer (c=10 M) 0.5 l
BigDye 3.1 Mix 1 l
ddH2O ad 10 l
PCR programme: