2 MATERIAL AND METHODS
2.1 Methods
2.1.4 Molecular biological methods
Organs were homogenized in TRI
ZOLReagent (Invitrogen) with a T8 basic Ultra Turrax.
200 l Chloroform per ml TRI
ZOLReagent were added and samples were shaken for 30 sec;
incubated for 5 min at room temperature and then centrifuged at 13000 rpm for 15 min at 4°C
in a microfuge. Subsequently, the aqueous phase was transferred to a new reaction tube and
500 l isopropanol, 120
l 5 M ammonium acetate and 10 l 0.5% (w/v) Glycogen wereadded per ml TRI
ZOLReagent. RNA was precipitated for 15 min at -20°C and sedimented
by centrifugation at 13000 rpm for 15 min at 4°C in a microfuge. The supernatant was
discarded and the pellet was washed with icecold 70% ethanol. The samples were centrifuged
at 8000 rpm for 5 min at 4°C in a microfuge; the supernatants were discarded and the pellet
was dried at the air. Then, the pellet was solved in deionized water and RNA concentration
and quality were determined with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
CA, USA).
Material and Methods 31
2.1.4.2 cDNA synthesis
2 g total RNA per sample were transcribed to cDNA with random hexamer primers and SuperScript II Reverse Transcriptase (Invitrogen according) according to manufacturer´s instructions.
2.1.4.3 Semi-quantitative Real-Time RT PCR
Semi-quantitative Real-Time RT PCR reactions contained 1x SYBR Green mix (Applied Biosystems, Foster City, CA, USA), 10 pmol forward-primer, 10 pmol reverse-primer and 5 l cDNA template (2.1.4.2) diluted 1:10 to 1:50 in UltraPure Water (Millipore) in a total volume of 30
l. The amplification was performed with an ABI Prism 7000 sequencedetection system (Applied Biosystems) with the following programm: 2 min 50°C, 10 min 95°C and 40 cycles with 15 sec 95°C and 15 sec 60°C. The runs were evaluated with the SDS2.2.2 Software (Applied Biosystems). Specificity of the products was assured by measurement of the products melting points. Amplification of PCR products derived from contaminating genomic DNA was excluded by the use of intron-exon spanning primer pairs (see 2.2.2). The expression of Ribosomal Protein Subunit 9 (RPS9) was used for standardization. The relative expression was calculated with the CT method. Relative expression values of proteasomal catalytic -subunits and POMP were determined from 3 mice per group and time point.
2.1.4.4 Molecular Cloning of vector constructs for retroviral transduction
The vector pDest-Super used for retroviral transduction of MEFs (2.1.2.2) was provided by
David Ermert (MPIIB). It is a hybrid plasmid, in which the MultiSite Gateway Three
Fragment recombination site from pDest R4-R3 (Invitrogen) substitutes the cloning site of
pSuper.retro.puro (OligoEngine, Seattle, WA, USA), which provides the flanking long
terminal repeats (LTR) of MSCV and puromycin resistance. DNA-sequences encoding 5 and
5i with C-terminal Flag-tag were amplified from a murine liver cDNA library (2.1.4.2) by
nested PCR with Expand High Fidelity Polymerase (Roche Applied Science). First, inserts
were amplified using primer pairs 5-start-for/ 5-Flag-rev and lmp7-start-for/lmp7-Flag-rev
(see 2.2.2) for 5- and 5i-Flag, respectively. The PCR products from the first reaction were
used for further amplification with attB1- and attB2-adapter primers. Inserts with an
IRES-eGFP sequence were amplified from a plasmid provided by Marcus Koch (MPIIB) with
primers IRES-eGFP-for/IRES-eGFP-rev (see 2.2.2). The inserts were purified by
electrophoresis on a 1% Agarose Gel in 1x TAE (40 mM Tris-acetate pH8.5, 2 mM EDTA)
and the QuiaQuick Gel Extration Kit (Quiagen, Düsseldorf, Germany) according to
manufacturer’s instructions. Purified 5- and 5i-Flag inserts were subcloned into the entry
vector pDONR221 (Invitrogen) and IRES-eGFP inserts into pDONR™P2R-P3 (Invitrogen) with the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual. The pDONR™P4-P1R-EF-1 entry vector containing the sequence of the EF-1-promoter was provided by David Ermert (MPIIB). Final expression vectors were generated by a LR-recombination reaction between pDest-Super, pDONR™P4-P1R-EF-1, pDONR221-5-Flag or pDONR221-5i-Flag and pDONR™P2R-P3-IRES-eGFP according to manufacturer´s instructions resulting in the expression vectors pEX-EF-1-5-Flag-IRES-eGFP and pEX-EF-1-5i-Flag-IRES-eGFP, respectively. One Shot TOP10 chemically competent E. coli (Invitrogen) were transformed with the plasmids according to manufacturer’s instructions. Ampicillin resistant, Chloramphenicol sensitive clones were verified as positive clones by colony PCR with primers 5-for/5-rev and 5i-for/5i-rev for pEX-EF-1-5-Flag-IRES-eGFP and pEX-EF-1-5i-Flag-IRES-eGFP, respectively. Verified clones were cultured and the plasmids were purified with the QuiaPrep Spin Miniprep Kit (Quiagen) according to manufacturer’s instructions.
2.1.4.5 Preparation of retroviral particles
The vector pEX-EF-1-eGFP, which was provided by David Ermert (MPIIB) expressing eGFP from the EF-1 promoter, was used as empty vector control in the following experiments. Packaging of the expression vectors 1-5-Flag-IRES-eGFP, pEX-EF-1-5i-Flag-IRES-eGFP and pEX-EF-1-eGFP into ecotrophic retroviral particles was achieved by transient transfection of Phoenix E cells (2.1.2.1) by Ca-phosphate precipitation.
2 x 10
6Phoenix E cells were seeded per cell culture dish with 10 cm-diameter. Next day, Ca-phosphate-DNA precipitates were generated by mixing 20
g plasmid DNA (2.1.4.4) with32 l 2 M CaCl
2and 300
l 2x HBS Buffer (Clontech, Saint-Germain-en-Laye, France)adjusted to a final volume of 600
l with deionized water. The mixture was incubated for30 min at room temperature and the resulting precipitates were cautiously dispersed on the 60-70% confluent monolayers of Phoenix E cells. 24 h later the medium was exchanged against fresh D10 medium. The supernatants containing retroviral particles were harvested 24 and 48 h later, filtered through a sterile filter with 0,45 m pore-size and polybrene was added to a final concentration of 4 g/ml.
2.1.4.6 Retroviral transduction of MEFs