El lenguaje WSML

2.4.1. RNA isolation

Total RNA was isolated from cells using the QIAamp RNA isolation kit following the manufacturer’s protocol (Qiagen, UK). Briefly, cells were lysed in the provided buffer and further homogenised by use of shredder columns provided. Homogenised cell lysates were then applied to a second column to which the total RNA was bound and impurities washed away. Total RNA was then eluted and re-suspended in RNase-free water.

2.4.2. Reverse transcription of total RNA to cDNA

Reverse transcription of total single-stranded RNA into complementary DNA (cDNA) was performed using an ABI High Capacity Kit according to the manufacturer’s instructions (Applied Biosystems, UK).

The 2x master mix for each reaction contained:

Reagent Volume (µl) reaction was then reverse transcribed using a thermal cycler with the following programme:

Step Temp (°C) Time (min)

1 25 10

2 37 120

3 85 5

4 4 ∞

The cDNA was diluted to 100 μl with nuclease-free H2O prior to real-time PCR.

2.4.3. Polymerase chain reaction (PCR)

The genes of interest, necessary for cloning into viral vectors, were amplified by PCR. For ZAP-70, the RNA was extracted from BALB/c and SKG splenocytes. The modified ΔNGFR coding sequenced was

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amplified from murine brain DNA kindly provided by Dr Alexander Annenkov (Bone and Joint Research Unit, Queen Mary, London, UK). PCR primers (described in section 2.1.3) were designed for ZAP-70 to create a 5’_BamHI site and either a BstBI or SpeI restriction site at the 3’ end. NGFR primers were designed to add a 5’_BspHI restriction site and to truncate the intra-cytoplasmic tail from residue 248 and add a NotI site. PCR reactions were carried out with the following PCR mix:

Reagent PCR mix using a Peltier Technology Thermal Cycler (PTC-200, MJ Research Inc.):

Step Temp (°C) Time

2.4.4. Assessment of DNA on agarose gels and DNA purification

Amplification of the correct size fragment was assessed by agarose gel electrophoresis. Thus, PCR products, as well as plasmid DNA, were mixed with appropriate volume of 6x bromophenol blue loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% v/v glycerol in TAE buffer). DNA was separated on a 1% agarose gel with 0.5x TAE buffer at 100 V. Ethidium bromide (0.5 μg/ml) was incorporated in the agarose gel to visualise DNA under ultraviolet light. The DNA products were then excised from the gel by cutting the agarose matrix, and further purified using the Qiaquick Gel Extraction Kit following manufacturer’s instructions (Quiagen, UK). Briefly, the gel fragment was

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dissolved in high salt buffer, which allows DNA binding to the silica membrane of the Qiaquick column. DNA was then eluted with low-salt buffer, or with water. Purified DNA products were verified by DNA sequencing analysis (Genome Center, Queen Mary, London, UK).

2.4.5. Quantitative PCR

Quantitative PCR (qPCR), also called real-time PCR, enables to monitor the progress of the PCR as it occurs since data is therefore collected throughout the PCR process, rather than at the end of the PCR. In qPCR, the reactions are characterised by the point in time during cycling when amplification of a target gene is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. qPCR reactions were carried out using ABI Taqman fluorogenic probes (detailed in section 2.1.5) in p384 microtitre plates and as follows:

Reagent qPCR mix Corbett Lifesciences Rotorgene 6000 thermocycler (Corbett Lifesciences, Sydney) or ABI AB7900HT 384 platform system (Applied Biosystems): control for variations in the absolute quantity of cDNA input, the Ct values of both the calibrator and the samples of interest were normalised to the level of an endogenous housekeeping gene, in this case Hprt1, which is assumed to remain at constant levels between samples.

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2.4.6. Cloning of bi-cistronic constructs

All DNA restriction enzymes were purchased from New England Biolabs Inc. (Hitchin, UK) unless otherwise stated. All DNA constructs were sequenced at the Genome Centre, Queen Mary (London, UK).

The self-inactivating lentiviral vector pHR’SIN.cPPT-SEW was kindly provided by Dr Y. Ikeda (Mayo Clinic, Rochester, MN, USA). I first cloned bi-cistronic constructs - containing a primary gene (wild-type or SKG ZAP-70) and a secondary gene (eGFP or ΔNGFR) downstream of an IRES sequence, and sub-cloned those constructs into the pHR’SIN.cPPT-SEW lentiviral vector.

In the second phase of the project, the bi-cistronic constructs were sub-cloned in murine Moloney leukaemia virus vector MFG, kindly provided by Dr Alexander Annenkov (Bone and Joint research Unit, Queen Mary, London, UK).

2.4.6.1. Digestion and Ligation of DNA

Following amplification of the genes of interest by PCR (section 2.5.3.), DNA products and plasmid vectors were digested with the appropriate restriction enzymes, and separated on 1% agarose gels and purified (section 2.4.4). The DNA fragment to be inserted was added in molar excess (5 to 10 folds) to vector DNA and ligated at 16°C overnight with T4 DNA ligase (200 units per reaction) in a final volume of 10 μl. made ice-cold 0.1 M MgCl2. Cells were sedimented again by centrifugation (1,500 g, 10 min, 4°C), and re-suspended in 50 ml of ice-cold 0.1 M CaCl2 and incubated on ice for 20 min. A final centrifugation step (15,000 g, 10 min, 4°C) was performed. Cells were re-suspended in 12.5 ml of ice-cold 0.1 M CaCl2 with 14% glycerol. Competent cells were aliquoted into Eppendorf tubes and snap-frozen in liquid nitrogen before storing at -70°C until future use.

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2.4.6.3. Transformation of E.coli

50 μl of competent DH5α E.coli cells and 3 μl of ligation reaction were incubated on ice for 30 min, heat-shocked for 2 min at 42°C, then grown in 5 ml LB medium at 37°C for 60 min with shaking, to allow recovery and plasmid gene expression. 200 µl of these cultures were plated onto LB Agar plates (1.5% w/v bactoagar) with ampicillin (100 µg/ml) and grown overnight at 37°C. Individual clones were picked and grown in 5 ml LB with ampicillin (100 μg/ml) at 37°C overnight with vigorous shaking.

2.4.6.4. Purification of Plasmid DNA

Bacterial cells were sedimented by centrifugation (11,000 g, 1 min, RT) and DNA was purified using the PureLink HiPure Plasmid Miniprep Kit following manufacturer’s instructions (Invitrogen, UK). The protocol is based on anion-exchange chromatography. Briefly, bacterial cells were re-suspended in the presence of RNase and lysed under high alkaline conditions to solubilise the cell membrane. Cells lysate was passed through a pre-packed anion exchange column and the negatively charged phosphates on the DNA backbone interacted with the positive charges on the surface of the resin.

RNA, proteins, carbohydrates and other impurities were washed away under moderate salt conditions, before elution of the plasmid DNA under high salt conditions.

A restriction analysis digest was carried out on each clone to verify the presence of the correct insert.

Appropriate clones were picked (1 ml of the 5 ml culture) for large-scale culture grown in 500 ml LB with ampicillin (100 µg/ml). DNA was purified using the PureLinK HiPure Plasmid Maxiprep Kit following manufacturer’s instructions (Invitrogen, UK). Plasmid DNA concentration was determined with Pharmacia GeneQuant spectrophotometer (Cambridge, UK) at 260nm. Correct cloning was confirmed by sequencing analysis.

2.4.6.5. Transient expression of protein

To verify that the bi-cistronic constructs cloned in viral vectors were functional, HEK 293T cells were transfected by calcium phosphate DNA co-precipitation (Chen and Okayama 1988) to transiently express the primary and secondary genes contained in the viral vector. Cells were seeded (2.5 x 10⁶ / 10 cm plate) the day before to achieve 60-80% confluence on the day of transfection. One hour before transfection, 25 µg/ml chloroquine (Sigma-Aldrich, UK) was added to each plate. The transfection mix was prepared as follows:

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Per 10 cm plate Volume / concentration

2x HBS 500 µl

DNA plasmid 20 µg DNA

2.5 M CaCl2 50 µl

dH2O Complete to 1 ml

The CaCl2 was added last to the transfection mix and very gradually with constant gentle shaking. The precipitation mix was left at RT for 20 min. The transfection mix was then added gradually to the cells and incubated at 37°C for 6h before replacing the chloroquine-containing medium with fresh DMEM complete medium. Cells were collected 2-4 days post-transfection for assessment of protein expression, either via flow cytometry (section 2.6.) or Western blotting (section 2.3.)