Temporada 6, capítulo 8 Lisa en los deportes.

3.4.1 pCAG-3SIP-based vectors (MCM, eGFP, xCT)

Plasmid pCAG-3SIP drives gene expression from a highly efficient CAG promoter and encodes an IRES-linked bicistronic sequence, including the puromycin N-

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acetyltransferase gene (purR) for stable selection. pCAG-3SIP was used for stable expression of the fusion protein MerCreMer, consisting of Cre recombinase (Cre) flanked by two mutated oestrogen receptors (Mer) (Verrou et al., 1999). The MerCreMer cassette was recovered from pBluescript-MerCreMer by EcoRI digestion

and cloned into EcoRI digested and dephosphorylated pCAG-3SIP, yielding pCAG-

3SIP-MerCreMer (figure 11). The insert orientation was controlled by analytic digestion with SalI and NheI/XhoI, respectively.

eGFP was isolated from pEGFP-N1 (Clontech, Saint-Germain-en-Laye, France) by

BamHI/XbaI digestion. The 751 bp fragment was blunted by Klenow fill-in reaction

and cloned into EcoRI digested, blunted and dephosphorylated pCAG-3SIP, yielding

pCAG-3SIP-eGFP (figure 11). The correct orientation of the insert was verified by

EcoRI/EcoRV and XmnI digestion, respectively.

pCAG-3SIP-xCT (figure 11) was provided by Ana Banjac, encoding the murine DNA sequence of xCT light chain (Banjac Ana, PhD thesis, 2005).

3.4.2 p442-PL1-based PHGPx expression vectors

The 3rd generation lentiviral vector p442-PL1 (kind gift from Tim Schröder, GSF, Munich, Germany) was used for efficient gene transfer and expression in MEFs by viral infection. Gene expression in p442-PL1 is regulated by an SFFV promoter, synthesizing a bicistronic mRNA sequence that is linked by an internal ribosome entry site (IRES). The fluorescence marker VENUSnucmem including a nuclear membrane anchor (nucmem) is encoded at the 3’ position of the bicistronic expression cassette. As described below, various genes were cloned into the 5’ position of the expression cassette, upstream of the IRES.

3.4.4 Cloning of tagged PHGPx

The PHGPx gene was amplified from murine testis cDNA by RT-PCR with primers “PHGPx complete for1” and “PHGPx complete rev1”. The purified 721 nucleotide product was cloned into pDrive according to manufacturers’ instructions (Qiagen PCR Cloning Kit). pDrive-PHGPx was used as template for a PCR with primers

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“PHGPx HAtag for1” and “PHGPx E4 rev1”, synthesizing an N-terminal HA-tagged fusion of PHGPx. The 351 bp PCR product was digested with BamHI and BssHII and

the 72 bp fragment was used to replace the N-terminal part of PHGPx in pDrive- PHGPx, yielding pDrive-HA-PHGPx. The pDrive-HA-PHGPx clones were tested by analytic restriction digestion with EcoRI and sequenced with primers ”T7 promoter”

and “SP6 promoter”. The HA-tagged PHGPx sequence was transferred from pDrive- HA-PHGPx to the lentiviral expression vector p442-PL1 by restriction digestion with

BamHI and XbaI. p442-HA-PHGPx clones (figure 11) were analyzed by restriction

digestion (EcoRI and BamHI/XbaI) and sequenced with primers ”5’-LTR for1” and

“IRES rev1”.

Figure 11: Schematic representation of all cloned expression vectors. Depicted are the pCAG-3SIP-

based vectors, containing an efficient CAG promoter and a PuroR cassette (puromycin N- acetyltransferase). The p442-PL1-based lentiviral vectors contain a SFFVp (spleen focus forming virus promoter) and a VENUSnucmem fluorescence marker. The cloned PHGPx contains an N-terminal HA-tag and FSH-tag (Flag-, Strep-, HA-tag), respectively. The two mutant forms of HA-PHGPx have a Cys (red asterisk) and Ser (green asterisk) instead of Sec.

For future studies, HA-PHGPx was additionally tagged with an optimised TAPe- (enhanced tandem affinity purification) tag, which has been developed and functionally tested for B-raf and C-Raf (Raf1) in Marius Ueffing´s laboratory at the GSF (kind gift from Dr. Johannes Gloeckner, GSF, Munich, Germany). This tag was specifically designed for the purification of native protein complexes from mammalian cells. The TAPe-tag facilitates rapid and native purification of protein complexes for subsequent identification of PHGPx binding partners by LC-MS/MS as well as functional assays. The combination of two medium-affinity binding epitopes (tandem

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Strep-tag II and a FLAG-tag) reduces the tag size to approximately 5 kDa. Hence, the N-terminal TAP-tag/PHGPx fusion protein is only marginally larger than wild type PHGPx. The N-terminal part of HA-PHGPx (lacking the start codon) was amplified with primers “HA-PHGPx for1” and “HA-PHGPx rev2”, introducing a 5’ NheI and a 3’ XhoI restriction site. The 349 bp PCR product was purified and cloned into pcDNA3-

NTAPe with NheI/XhoI. The N-terminal part of PHGPx, now including the TAPe-tag,

was replaced in pDrive-HA-PHGPx by BamHI/BssHII, yielding a Flag, Strep (2x), and

HA-tagged PHGPx gene, designated pDrive-FSH-PHGPx (figure 11). The constructs were analysed by restriction digestion with BamHI/BglII and sequenced with primers

”T7 promoter” and “SP6 promoter”. The FSH-PHGPx sequence was transferred to p442-PL1 with BamHI/XbaI as described above, yielding p442-FSH-PHGPx.

3.4.5 PHGPx Mutagenesis

Mutations of single amino acids within PHGPx were performed by PCR mutagenesis with pDrive-HA-PHGPx in a volume of 50 µl. Primers of 33 nucleotides were designed, such that the primer sequence consists of a mutated codon, flanked by wild type sequences of at least 10 nucleotides. Mutated codons were chosen according to the highest murine codon usage of the respective amino acid. The Sec codon (UGA) at position 46 of murine PHGPx was mutated to Cys (UGC) and Ser (AGC) with primers “PHGPx UC for1/rev1” and “PHGPx US for1/rev1”, respectively. The PCR-reactions were performed with reduced primer annealing temperature (- 6°C), due to the mismatch of primer and wild type PHGPx sequence. Prior to the transformation of competent cells, PCR reactions were digested with DpnI for 60 min

to remove methylated template DNA. Mutations were verified by sequencing in pDrive with primers ”T7 promoter” and “SP6 promoter”. The mutated sequences were transferred to the lentiviral expression vector p442-PL1 as described above, yielding p442-HA-PHGPx-UC and p442-HA-PHGPx-US, respectively (figure11).

3.4.5 Bcl-2 expression vectors

The sequence for wild type Bcl-2 was recovered from pBabe-Bcl-2-puro (kind gift from Gerard Evan, London, UK; (Fanidi et al., 1992)) by restriction digestion with

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dephosphorylated vector p442-PL1, yielding p442-Bcl-2 (figure 11). Single clones were checked for correct orientation of the insert by restriction digestion with BamHI

and XmaI, respectively.

Plasmid pIRES-neo3-Bcl-2-ActA is based on the CMV promoter-driven expression vector pIRESneo3 (Clontech), containing an IRES and a neomycin resistance cassette (NeoR) for stable selection of expressing cell clones with G418. The plasmid encodes a mutated Bcl-2 in which the membrane anchor was replaced by the mitochondrial insertion sequence of ActA (Bcl-2-ActA) (Zhu et al., 1996), targeting Bcl-2 localisation to the outer mitochondrial membrane. Plasmid pIRES-neo3-Bcl-2- ActA was a kind gift from Prof. Peter Daniel (University Medical Centre Charité, Berlin-Buch, Germany).