Porcine adenovirus type 3 genome

A method for obtaining a recombinant PAV-3 comprising a heterologous nucleotide sequence inserted into a PAV-3 insertion site located in a region selected from the group consisting of the E1 region, and the E4 region of the PAV-3 genome, the method comprising the steps of:

• (a) providing a PAV-3 genome capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:
• 1;
• (b) providing a heterologous nucleotide sequence;
• (c) linking the heterologous nucleotide sequence to guide sequences, the guide sequences being capable of duplex formation under conditions of high stringency to said PAV-3 genome sequences flanking the PAV-3 insertion site, or the complement of said sequences, such that guide sequences are present at both ends of the heterologous sequence;
• (d) introducing the construct from step (c) into a cell together with the PAV-3 genome;
• (e) allowing homologous recombination to occur between the two sequences from step (d) to generate a recombinant PAV-3 genome;
• (f) purifying the recombinant PAV-3 genome;
• (g) inserting the recombinant PAV-3 genome into a mammalian cell;
• (h) culturing the mammalian cell under conditions wherein the recombinant PAV-3 genome is replicated and packaged;
• and
• (i) optionally collecting the recombinant PAV-3 from the cell or the culture medium.

A method for obtaining a full-length genomic clone of a PAV-3 genome, the method comprising:

• (a) providing two or more cloned segments of the PAV-3 genome, wherein said segments are capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof, and wherein the cloned segments of the PAV-3 genome, taken together, represent the entire PAV-3 genome;
• (b) introducing the two or more cloned segments of the PAV-3 genome into a cell;
• (c) allowing homologous recombination to occur within the cell between the two or more cloned segments of the PAV-3 genome to generate a full-length PAV-3 genome;
• and
• (d) optionally purifying the full-length PAV-3 genome from the cell.

A method for obtaining a recombinant PAV-3 comprising a heterologous nucleotide sequence inserted into a PAV-3 insertion site located in a region selected from the group consisting of the E1 region and the E4 region of the PAV-3 genome, comprising the steps of

• a) introducing a recombinant plasmid into a host cell in combination with a PAV-3 genome, wherein said plasmid comprises a heterologous nucleotide sequence flanked by nucleotide sequences that are capable of duplex formation under conditions of high stringency to PAV-3 nucleotide sequences flanking the PAV-3 insertion site, or the complement of said sequences, and wherein the PAV-3 genome is capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof,
• b) allowing homologous recombination to occur between the plasmid and the PAV-3 genome thereby generating a recombinant PAV-3 comprising said heterologous nucleotide sequences;
• c) isolating said recombinant PAV-3;
• d) introducing said isolated PAV-3 into a mammalian cell permissive for growth of said PAV-3;
• e) culturing said mammalian cell under conditions suitable for PAV-3 replication and packaging;
• and
• f) optionally, collecting said recombinant PAV-3 produced from step e).