Improved methods for producing polynucleotide libraries in vaccinia virus/eukaryotic cells

A method of constructing a library comprising a plurality of polynucleotides of interest, comprising:

• (a) cleaving an isolated poxvirus genome to produce a first viral fragment and a second viral fragment, wherein the first fragment is nonhomologous with the second fragment;
• (b) providing a population of transfer plasmids each comprising a polynucleotide of interest flanked by a 5′ flanking region and a 3′ flanking region, wherein the 5′ flanking region comprises a region homologous to the 3′ end of the first viral fragment and the 3′ flanking region comprises a region homologous to the 5′ end of the second viral fragment;
• and wherein the transfer plasmids are capable of homologous recombination with the first and second viral fragments such that a viable poxvirus genome is formed;
• (c) introducing the transfer plasmids and the first and second viral fragments into a mammalian host cell permissive for poxvirus infectivity;
• (d) adding an inhibitor of poxvirus assembly;
• and
• (e) allowing the transfer plasmid and the first and second viral fragments to undergo homologous recombination, thereby producing a library of viable modified poxvirus genomes each comprising a heterologous nucleic acid.