Consolidación del Teatro Experimental Universitario

Electroporation of MEFs

Logarithmically growing mammalian cells were harvested by trypsinization and washed in PBS. Approximately 3x106 _{cells in 500 μl PBS were used per}

electroporation. The cells were mixed with 20-30 μg of plasmid DNA in 0.4 cm cuvettes and electroporated using the GenePulser II apparatus (Bio Rad, Munich, Germany) with standard settings of 240 Volt and 950 μF capacitance. The transfected cells were seeded on a 10 cm cell culture dish in standard DMEM medium with 10% FCS. The selection of transfected cells was initiated 24 h after electroporation with the appropriate selection marker (puromycin (up to 2 µg/ml) or hygromycin B (up to 300 µg/ml)), with gradually increasing doses. Stably transfected transgene-expressing cells lines were obtained after long term selection over suitable selection markers.

Transduction of cells using lentiviruses

Lentiviral-transduction is a fast and efficient method of gene delivery into cells. Due to the high infection rates of even non-dividing and hardly transfectable cells, the absence of adverse effects on target cells, and the stable transgene expression after provirus integration, this method was used to transduce primary MEFs. The HIV- based 3rd _{generation ecotropic lentiviral vector system was used for the in vitro}

delivery of genes of interest into different mouse cell lines. In order to avoid any recombination events, all necessary proteins for lentivirus production including Env, Gag, Pol and Rev are encoded by three different plasmids. To ensure maximum bio- safety, the natural promoter is replaced by promoters from other viruses (e.g. Rous sarcoma virus), the 5´and 3´-LTRs are truncated, all the accessory genes are removed, and the virus is pseudotyped with glycoproteins from other viruses.

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Lentiviral vector system and their modifications

The lentiviral vector system consists of the following components: Packaging vectors: 391 K73 pEcoEnv-IRES-puro 392 pRSV_Rev 393 pMDLg_pRRE Transfer vectors: 441 pRRL.PPT.SF.IRES-golgiVENUS 442 pRRL.PPT.SF.IRES-VENUSnucmem 443 pRRL.PPT.SF.IRES-mitoVENUS

The aforementioned transfer vectors 441, 442, and 443 were modified by deleting the EcoRI and XhoI restriction sites to generate pre-linker (PL) intermediates. These were digested with BamHI and SnaBI. Subsequently, the lenti-linker 1 was inserted that contains multiple unique restriction sites to generate the 441 L1, 442 L1 and 443 L1 transfer vectors. The puromycin acetyltransferase gene was amplified by PCR from pHC-BZ-SVpuro. SnaBI and BsrG1 sites were attached to the ends of the PCR product by the primers used for amplification. The SnaB1and BsrGI digested PCR fragment was cloned into the MscI- and BsrGI-digested 442 L1 nucmembVENUS plasmid to generate the 442 L1 IRES Puro vector (Figure 10).

Cloning of the Ha-ras and c-myc oncogenes into the lentiviral vectors

pBJ3Ωc-myc and pUC EJ6.6 Ha-rasV12 _{were obtained from Dr. Hartmut Land. A 1.7}

kb fragment covering the mutated (Val12) Ha-ras coding sequence was amplified by PCR using pUC EJ6.6 Ha-rasV12 _{as template. Two EcoRI sites were introduced by}

the primers used for amplification. Then, the 1.7 kb fragment was digested with EcoRI and cloned into the same site of 441 L1 golgiVENUS to generate 441 L1 Ha- rasV12 _{golgiVENUS. The pBJ3Ω c-myc vector was digested with Ecl136II and an}

EcoRI linker (New England Biolabs GmbH, Frankfurt, Germany) was inserted. Then, the plasmid was digested with EcoRI and the excised 1.3 kb fragment containing the

Materials and Methods

c-myc coding sequence was cloned into the 443 L1 mitoVENUS plasmid to generate 443 L1 c-myc mitoVENUS.

Figure 10. Schematic representation of the HIV-based lentiviral vector. The transfer vector carries the gene of interest and consists of the truncated genome of the virus that is needed for infection and integration. The transgene is expressed from the SFFV promoter. By incorporating the sequence for an internal ribosomal entry site (IRES), bicistronic messages are created. RSV: Rous sarcoma virus promoter, PPT: polypurine tract, SFFV: spleen foci forming virus, pac: puromycin acetyltransferase (resistance gene), PRE: Post-transcriptional regulatory element, RRE: Rev-responsive element, SIN: self-inactivating. Several restriction sites are incorporated into the lenti-linker 1 to facilitate the insertion of the gene of interest.

Cloning of mouse thioredoxin reductase 1 gene into the lentiviral vector

Mouse Txnrd1 (transcript variant 2 or 2ATG, Accession number: NM_001042513.1) containing the SECIS element was amplified from brain cDNA by PCR using Pfx DNA polymerase and the following primers: Oligo-Nhe1-Txnrd1 for1 (5’- TGATGCTAGCAATGGCTCCAAAGATCCCCCTG-3’) and Oligo-Xba1-Txnrd1 rev (5’-ACGTCTAGACAGGAAAAGGTTGAGCTCAACAG-3´). This PCR product was cloned into the pDrive vector using the PCR cloning kit (Qiagen, Hilden, Germany) generating pDrive-Txnrd1 wt (2ATG). The sequencing of the amplified product revealed a T to C nucleotide exchange in the non-coding SECIS sequence, which was reverted by site-directed mutagenesis. Subsequently, the plasmid pDrive-Txnrd1 was digested with NheI and OliI, the Txnrd1 fragment isolated and cloned into the pcDNA3.1 SF-TAP ASA vector, which was generated by site-directed mutagenesis from pcDNA3.1 SF-TAP. The pcDNA3.1 SF-TAP ASA vector was digested with XhoI,

Materials and Methods

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treated with Klenow enzyme, and then digested with NheI. The NheI/OliI-digested Txnrd1 fragment was cloned into this vector to generate pSF-Txnrd1. From this plasmid, the Txnrd1 fragment was cloned as an EcoRI-XbaI fragment into the 442 L1 IRES-Puro (Puro = puromycin acetyltransferase) plasmid to generate 442 L1 SF- Txnrd1 wt (2ATG) IRES-Puro.

Site-directed mutagensis of Txnrd1

Thioredoxin reductase1 has two catalytic centers: one at the N-terminus containing 2 cysteine residues (C59 and C64) and the second at the C-terminus formed by cysteine and selenocysteine (C497 and U498). All three cysteine residues and the Sec were individually mutated to serine and cysteine, respectively. The following mutants were created by site-directed mutagenesis using the respective pairs of oligonucleotides. The mutated bases are depicted in red.

wt (N-terminal) GGA GGA ACG TGT GTG AAT GTG GGT TGC ATA C59S GGA GGA ACG TCC GTG AAT GTG GGT TGC ATA

C64S GGA GGA ACG TGT GTG AAT GTG GGT TCC ATA

wt (C-terminus) CTC CAG TCT GGC TGC TGA GGT TAA GCC CC C497S CTC CAG TCT GGC TCC TGA GGT TAA GCC CC

U498C CTC CAG TCT GGC TGC TGC GGT TAA GCC CC

U498S CTC CAG TCT GGC TGC TCC GGT TAA GCC CC

U498STOP CTC CAG TCT GGC TGC TAA GGT TAA GCC CC

All mutations were carried out with the pDrive-Txnrd1 wt (2ATG) plasmid. From these plasmids, the different C-terminal mutant DNA fragments were isolated with Eco47III and XbaI and inserted into the 442 L1 SF-Txnrd1 wt (2ATG) IRES Puro plasmid digested with the same enzymes. For generating the N-terminal mutants (C59S, C64S and SS), the BclI and Eco47III restriction enzymes were used to exchange the wild-type for the mutated sequences.

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Lentiviral production and transduction of target cells

HEK 293-T cells were used for virus production. 5x106 _{HEK 293-T cells were seeded}

in a 10 cm cell culture dish and incubated for 12 h prior to transfection. Cells were grown to 70% confluency and then used for transfection. The packaging cell line was transfected simultaneously with four plasmids by the calcium phosphate method.

A typical transfection mix for one 10 cm plate consisted of: 2 μg pEcoEnv-IRES-puro

5 μg pMDLg_pRRE 10 μg pRSV_Rev 5 μg transfer vectors

The above vectors were mixed with 50 µl of 2.5 M calcium chloride in water to a final volume of 500 µl. To this mixture, 500 µl of 2 x HeBS (HEPES buffered saline, pH 7.5) was added while air was bubbled through the mixture and then kept for 20 min at room temperature. 10 ml of transfection medium containing 25 μM chloroquine was added to the cell monolayer. 1 ml of the transfection mix was added to each plate and the cells were incubated for 8-12 h. After incubation, the transfection medium was replaced with 8 ml of fresh transfection medium without chloroquine and the cells were incubated for further 36 h (Figure 11). The supernatant containing the virus particles was collected after 36 h, filtrated through a 0.22 µm sterile filter, and concentrated by ultracentrifugation at 8000 rpm for at least 16 h. The virus pellet was resuspended in 200 µl of cell culture medium and stored at -80°C until further use. For transduction, 1x105 _{MEFs were infected with 10 µl of virus suspension for 24 h.}

The transduction efficiency was analyzed by FACS 48 h thereafter by monitoring VENUS expression (in case of 441 L1 Ha-rasV12 _{IRES golgiVENUS and 443 L1 c-}

myc IRES mitoVENUS). Stable cell lines were generated by transducing the cells with viruses carrying the puromycin resistance gene (for 442 L1 IRES-Puro) and subsequently selected with puromycin, starting form 0.5 µg/ml to the final concentration of 2 µg/ml.

Chloroquine (1000 x): 25 mM chloroquine in PBS

Materials and Methods

HeBS (2 x): 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.05 adjusted with NaOH

TF medium: Standard DMEM, 20 mM HEPES

Figure 11. Lentivirus production in packaging cell lines. All four plasmids were co-transfected in HEK 293-T cells using the calcium phosphate method. The supernatant was collected after 36 h. The virus particles were recovered by ultra-centrifugation and then used for the transduction of target cells.

env: gene encoding for the viral envelope, gag: gene coding for the viral glycoprotein, rev: gene

encoding the reverse transcriptase, goi: gene of interest.