Construction of E1-Substituted Adenovirus Vectors

Step 1: Insertion of gene of interest into shuttle plasmid

• Introduce the expression cassette into the multiple cloning site of pE1.1, downstream from the adenovirus packaging signal.

pE1.1 contains the first 353 nucleotides from the Ad5 genome, including the left Inverted Terminal Repeat (ITR) and the packaging signal y. These sequences are flanked by two rare-cutting enzymes (PacI and SwaI) on the left side, and a multiple cloning site on the right side. The plasmid contains in addition a cos site for DNA packaging into phage l, a pUC19-derived origin of replication, and a kanamycin-resistance gene. The PflMI site originally present in kanamycin-resistance gene (Tn903) has been mutated.

• Digest the resulting plasmid with AlwNI, BstAPI, DraIII, or PflMI, whichever is not present in the expression cassette.

The recognition sites of these four restriction enzymes are interrupted palindromes. Both sets of sites (the one located at the end of the kanamycin-resistance gene and the one located next to the multiple cloning site) generate sticky ends which are not compatible with each other.

• Purify the fragment containing the expression cassette on agarose gel.

Step 2: Reconstitution of the Recombinant Adenovirus Genome in a Plasmid or Cosmid

• Ligate the purified fragment with SfiI-digested AdenoQuick™.

AdenoQuick™1.1 is a 39 kb plasmid that contains the right end of the Ad5 genome (nt 3504-end). The right ITR is flanked by PacI and SwaI sites. The two SfiI sites present in WT Ad5 DNA have been mutated. Two new SfiI sites have been inserted in place of the E1 region. The enzyme SfiI recognizes an interrupted palindrome. Both sites have been designed to generate different sticky ends, which are compatible with those generated at the AlwNI, BstAPI, DraIII and PflMI sites present in plasmid pE1.1. AdenoQuick™1.1 contains an ampicillin-resistance gene, a pUC19 origin of replication, and sequences from phage l as stuffer DNA. AdenoQuick™1.1 can be used to construct adenoviral vectors carrying inserts in the E1 region, with a maximal size of 5.1 kb. A derivative of AdenoQuick™1.1 (AdenoQuick™1.2) was constructed by deleting a 2.7 kb fragment from the E3 region. AdenoQuick™1.2 allows to construct adenoviral vectors carrying inserts in the E1 region, with a maximal size of about 7.8 kb.

• Package the ligated DNA into phage l, infect E. coli, and select Kan^r clones. Alternatively, transform E. coli directly by electroporation.

Using the l packaging extract from Stratagene, dozens to hundreds of clones are usually obtained.

• Check the resulting cosmid by restriction analysis (necessary if electroporation was used, optional at this time if packaging into phage l was used).

So far hundreds of recombinant clones obtained via packaging into l have been analyzed by restriction digest. All contained the expression cassettes inserted in correct orientation. Seventy percent of the clones obtained by electroporation were correct.

• Purify the cosmid (50 ml LB + Kan).

Cosmid DNA can be purified using anion-exchange columns (Nucleobond, Concert or Qiagen), silica-based columns (Promega Wizard), or CsCl gradient centrifugation. One ml bacterial culture can yield up to 3 mg DNA.

• Digest the cosmid with PacI or SwaI, whichever is not present in the expression cassette.

Step 3: Virus Recovery

• Transfect the linearized cosmid into helper cells (e.g. 293), using the CaPO₄ method or lipofection.
• Overlay the cell monolayer with agar and harvest a few plaques.

Plaques appear usually 7 to 10 days after transfection, sometimes as early as 4 days.

• - Perform a second plaque assay and analyze a few plaques to check the identity of the virus.

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Construction of E1- and E3-Substituted Adenovirus Vectors

Step 1: Insertion of genes of interest into shuttle plasmids

• Introduce the expression cassettes into the multiple cloning sites of pE1.2 (E1 region) and pE3.1 (E3 region).

pE1.2 contains the first 353 nucleotides of the Ad5 genome, preceded by PacI and SwaI sites, and followed by a multiple cloning site. It contains in addition a kanamycin-resistance gene and a pUC19-derived origin of replication. pE3.1 contains a l cos site of minimal size (180 bp), a multiple cloning site and a kanamycin-resistance gene.

• Digest the resulting plasmids with AlwNI, BstAPI, DraIII, or PflMI, whichever is not present in the expression cassettes.

As for pE1.1, the cutting sites of these enzymes were designed to generate different sticky ends (see color code).

• Purify the fragments containing the expression cassettes on agarose gel.

Step 2: Reconstitution of the Recombinant Adenovirus Genome in a Cosmid

• Ligate the purified fragments with SfiI-digested AdenoQuick™13.1.

AdenoQuick™13.1 is a 37 kb plasmid that contains the right end of the Ad5 genome (nt 3504-end), with a 2.7 kb deletion in the E3 region. Four SfiI sites have been inserted - two in the E1 region, and two in the E3 region, that generate four different sticky ends compatible with the AlwNI, BstAPI, DraIII and PflMI sites present in the intermediate plasmids pE1.2 and pE3.1. Taken together, the size of both expression cassettes should not exceed 7.6 kb.

• Package the ligated DNA into phage l, infect E. coli, and select Amp^r clones.

Although this ligation involves four DNA fragments, the efficiency is still very high and hundreds of clones can be obtained in a single experiment.

• Check the resulting cosmid by restriction analysis (optional at this time).
• Purify the cosmid DNA (50 ml LB + Kan).
• Digest the cosmid with PacI or SwaI, whichever is not present in the expression cassette.

Step 3: Virus Recovery

• Transfect the linearized cosmid into helper cells.
• Overlay the transfected cells with agar and harvest a few plaques.
• Perform a second plaque assay and analyze a few plaques to make sure of the stability of the recombinant virus.