Generic Molecular techniques

In order to start the construction o f a vector for any gene transfer protocol, a number of molecular techniques are required in the cloning steps for the production of suitable plasmid vectors, either for use as vectors themselves or for use later in viral vector preparations.

Techniques acquired for Cloning of Vectors

3.8.1 Restriction Digests

These were required to manipulate various cDNA sequences from the provided plasmids i.e. from other investigators and the ATCC. The plasmid map was studied and the appropriate restriction sites identified. Then, under conditions as dictated by the individual restriction endonucleases, restriction digests were carried out. The endonucleases were supplied by Boeringher Mannheim, GIBCO, or Promega. Reactions were typically carried out using lOU o f enzyme in a 20pl reaction with the relevant buffer. In any one reaction usually about l-2pg of DNA was digested.

3.8.2 Ligation Reactions

Reactions were set up in 20pl volumes containing approximately 10 ng o f vector and 50-200ng o f insert in Ix ligation buffer (10 mM rATP, 1 M Tris -HCL pH 7.4, 1 M MgCl] and 0.3 M DTT). One pi o f T4 DNA ligase (lU Boeringer) was added, the

reaction mixture made up to 20pl with sterile distilled water (SDW) and the reaction was carried out ovemightat 16° C.

3.8.3 Alkaline phosphatase reaction

This reaction was carried out to dephosphorylate the vector in order to prevent re ligation of linearised DNA molecules during cloning procedures. Briefly, calf intestinal phosphatase (CIP) was used to remove the 5' terminal phosphate group of the digested DNA fragments. One pi of CIP was added to the restriction digest mixture, and incubated for 1 hour at 37°C. Then, 5mM EDTA pH 8 was added and the mixture heated to 65°C for 20 minute to inactivate the CIP. The DNA was then phenol chloroform extracted. The aqueous phase containing the DNA was subsequently ethanol precipitated and resupended in TE 20pl.

3.8.4 Agarose gel electrophoresis

Agarose gel o f the various required percentages were made by dissolving Agarose in either IX Tris-borate (TBE) buffer (90mM Tris pH 8, 90mM orothoboric acid, 2.5mM EDTA) or Ix Tris acetate (TAE) buffer (x 50 buffer contains 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA (pH 8.0) made up to 1 litre with de­ ionised water). To visualise DNA, ethidium bromide was added at a final concentration o f 0.5 pg/ml. The gel was the then poured into a running tray and a comb was added to the tray, which was then removed when wells formed prior to the DNA samples being added. Once the tape and comb were removed the, appropriate buffer x l was added (depending upon what the gel was made fi"om), 10 ^1 o f sample was mixed with 5 gl o f x 6 loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF and 40%, (w/v) sucrose in water). An appropriate molecular weight marker was used to determine the size o f the DNA fragments. Typically, 0.7% w/v agarose was used for separation of fi*agment sizes between 0.8Kb-12Kb, whereas 0.5% w/v agarose separated between 0.1Kb-30Kb and 1.5% w/v between 0.2Kb-3Kb. The gels were run at 45-55mA and the negatively charged DNA then migrated towards the anode. The DNA was visualised using a UV transilluminator and photographed with a Polaroid black and white camera.

3.8.5 Bacterial transformations

A lOOpl o f competent cells (Epicurian Coll SURE 2 supercompetent Cells, Stratagene) were taken and thawed on ice in an eppendorf tube. In a volume o f 50 pi, a maximum amount o f 50 ng o f DNA taken and mixed with the competent cells and left on ice for a maximum o f 30 minutes. In a 42°C water bath, the cells were then heated shocked for 90seconds and then placed on ice for 2 minutes. 900 pi of LB SoC medium (50mls LB medium, 0.5mls IM MgSo4, 0.1 mis 20% Glucose) was added to the cells and the mixture was incubated for 1 hour at 37 ° C. The cells were then spun down and streaked out over LB/Ampicillin (Amp) plates. LB/Amp plates were made up from a autoclaved mixture of: 500ml de-ionised water, 5g tryptone, 2.5g yeast extract, 5g NaCl, 7.5g Agar. The mixture was allowed to cool, and ampicillin was added at a final concentration o f 50pg/ml. The mixture was the added to 90 mm petri dishes and allowed to set. The streaked out bacteria were the incubated at 37°C overnight.

3.8.6 Minipreps: (Requiredfor Colony Screens o f cloning vectors)

The petri dishes were removed from the incubator and a discrete colony was picked off the plate using a sterile toothpick and aseptic technique, and inoculated into 4mls LB/Amp. The mixture was placed in a shaker at 225 rpm overnight at 37°C. 1.5 mis of the mixture was centrifuged and the supernatant removed. The pellet was resuspended into lOOpl o f solution I (43.75ml H2O, 1.25ml Tris Stock IM, 5ml lOOmM EDTA, 0.45g glucose) and resuspended. Then 200 pi o f solution II (lOOpl 10% SDS, 20pl

lOM NaOH, 880pl H2O) was added to the mixture, inverting several times.

150pl o f solution III (30 ml 5M Potaasium acetate, 5.75ml Glacial acetic acid, 14.25ml H2O) was added, and the solution was shaken vigorously. The mixture was centrifuged in a microfuge (1300rpm) for 2 minutes. The supernatant was removed to a tube containing 400pl phenol/chloroform. The tube was shaken vigorously for 1 minute and then centrifuged again for 2 minutes. The supernatant was removed and added to a tube containing 200 pi o f chloroform and centrifuged for 2 minutes. The supernatant was removed and added to 800pl o f ethanol 100%, vortexed and

centrifuged for 2 minutes. The ethanol was removed and the pellet washed in 70% ethanol. The ethanol was removed and the pellet left to air diy and finally resupended in TE.

3.8.7 Maxiprep (for large scale production o f DNA)

Once the DNA was deemed to be correct, a sample was then grown up by maxipreping according to the Quiagen protocol. 150 mis of grown culture was taken and spun down to create a bacterial pellet, which was resupended in lOmls o f buffer PI (43.75mls H2O, 1.25mls Tris Stock IM, 5mls lOOmM EDTA, 0.45g glucose). The bacteria were completely redissolved and lOmls of buffer P2 (lOOpl 10% SDS, 20pl lOM NaOH, 880pl H2O) were added and incubated at room temperature for 5 minutes. The solution was mixed thoroughly by inverting the tube 5-6 times. Then 10 mis o f buffer P3 (30 ml 5M Potaasium acetate, 5.75ml Glacial acetic acid, 14.25ml H2O) was added and the solution mixed by inverting 5-6 times. The solution was then incubated on ice for 20 minutes. After incubation, the sample was mixed again before centrifugation was carried out at 15000 RPM for 30 minutes at 4°C.

After centrifugation the supernatant was removed promptly and applied over a Qiagen tip 500 which had been equilibrated by applying 10 mis of buffer QBT (750mM NaCl, 50 mM MOPS, pH 7.0, 15% isopropranol, 0.15% Triton X-100. The supernatant was applied and allowed to drain through the column by gravity flow. Once drained, the column was washed twice with 30 mis o f buffer QC (l.OM NaCl, 50mM MOPS, pH 7.0, 15% isopropranol). The DNA was then eluted off the column with 15 mis o f buffer QF (1.25M NaCl, 50mM Tris Cl, pH 8.5, 15% isopropranol). The DNA was then precipitated with 0.7 volumes o f isopropanol previously equilibrated to room temperature. The mixture was centrifuged immediately at > 15000g at 4°C for 30 minutes and the supernatant carefully removed. The DNA was washed in 15 mis o f cold 70 % ethanol and redissolved in TE. The yield o f DNA was calculated by measuring the DNA concentration on a UV spectrophotometer at 260nm.

3,8.8 Transfections

For the transfection, the cationic lipid lipofectamine was used. Lipofectamine interacts with DNA to form a lipid DNA complex. The resulting lipid - DNA complex fuses with the tissue culture cells under test and results in uptake and expression o f the DNA to the target cells.