Construction and Purification of Recombinant Ad Vector

The cassette cosmid for constructing recombinant Ad of the E1-substitution type, pAxCAwt is an 11-kb charomid vector bearing an Ad5 genome spanning map units (mu) 0-99.3 with deletions of E1 (mu 1.3-9.3) and E3 (mu 79.684.8) (24). The vector harbors the CAG promoter, which is a chicken p-actin

Fig. 1. K-ras expression plasmids. A 347-bp K-ras cDNA fragment containing ex-ons 1 and 2 and part of exon 3 is subcloned into the LNSX plasmid in antisense and sense orientation. LTR, long terminal repeat; SV, SV40 early promoter and enhancer; neo, bacterial neomycin phosphotransferase.

promoter fused to a cytomegalovirus enhancer (25,26), a unique Swal site, and a rabbit P-globin poly (A) sequence in the leftward orientation at the AdE1-deleted position (Adenovirus expression vector kit; TaKaRa).

3.2.1. Construction of Ad Vector

1. Digest and insert the Swal sites of the cloned K-ras fragment (spanning from nt 171 to 517) at the ends of the upstream and downstream primers into pAxCAwt (TaKaRa) in antisense (pAxCA-AS-K-ras) or sense orientation (pAxCA-S-K-ras) as described in Subheading 3.1.

2. Prepare the EcoT22l-digested adenoviral DNA-terminal protein complex (DNA-TPC) from the parent Ad5-dlX Ad, which has an E3 deletion (mu 79.6-84.8) (TakaRa) (27).

3. To construct the recombinant Ad, cotransfect 1 ^g of Ad5-dlX DNA-TPC digested with EcoT221 into 293 cells in a 60-mm dish together with 8 ^g of pAxCA-AS-K-ras or pAxCA-S-K-ras by the calcium phosphate method (Cell-Phect Transfection Kit; Amersham Pharmacia Biotech). The desired recombinant Ads are generated from overlapping recombination in the 293 cells.

4. One day later, spread the cells in three 96-well plates at a 10-fold serial dilution mixed with untransfected 293 cells.

5. After maintaining in culture for 10-15 d, further isolate and propagate the virus clones to assess restriction analysis. The resulting Ad vectors expressing the K-ras cDNA fragments in the antisense or sense orientation are designated AxCA-AS-K-ras and AxCA-S-K-ras, respectively.

3.2.2. Purification and Storage of Recombinant Ads

1. Use viral lysates of AxCA-AS-K-ras or AxCA-S-K-ras to infect 20-30 tissue culture dishes (150 mm) of 293 cells.

2. After 3-5 d, when they show a cytopathic effect, harvest the cells and then centrifuge at 220g for 5 min. Remove the medium and add 20-30 mL of 10 mM Tris-HCl, pH 8.0. After four cycles of freezing and thawing, spin the viral lysates at 2500g for 7 min and collect the Ad-containing supernatant.

3. Overlay the supernatant on the first CsCl gradient (10 mL of light CsCl and 10 mL of heavy CsCl solutions) in 1 x 3.5 in. Ultra-Clear tubes, and centrifuge at 20,000 rpm for 2 h at 4°C in an SW28 rotor (Beckman).

4. Collect the visible virus band and then overlay on the second CsCl gradient (4 mL of heavy CsCl and 4 mL of light CsCl solutions) in 9/16 x 3.5 in. Ultra-Clear tubes, and centrifuged at 20,000 rpm for 12-18 h at 4°C in an SW41 rotor (Beckman). Collect a sharp band consisting of virus particles.

5. Purify the viruses through a Bio-Gel P-6 DG chromatography column (EconoPac 10DG; Bio-Rad), and store at -80°C in PBS containing 13% glycerol.

3.2.3. Determination of Virus Titer

The infectivity of recombinant viruses, or titers of virus stocks, is determined by an end-point cytopathic effect assay (24).

1. Dispense 50 yL of DMEM with 10% FCS into each well of a 96-well plate, and then prepare eight rows of threefold serial dilution of the virus starting from a 10-4 dilution.

2. Add 3 x 105 293 cells in 50 yL of DMEM with 5% FCS to each well.

3. Incubate the plates at 37°C in 5% CO2 in air, and add 50 yL of DMEM with 10% FCS to each well every 3 d.

4. Twelve days later, determine by microscopy, the highest dilution (end point) showing the cytopathic effect, and calculate a 50% tissue culture infectious dose (TCID50). The TCID50/mL corresponds approx to 1 plaque-forming unit (PFU)/mL.

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