Methods of producing a library and methods of selecting polynucleotides of interest

Abstract

The present invention relates to a high efficiency method of introducing DNA into linear DNA viruses such as poxvirus, a method of producing libraries in linear DNA viruses such as poxvirus, and methods of selecting polynucleotides of interest based on cell nonviability or other phenotypes.

1 - 137 . (canceled)  
     
     
         138 . A method of constructing a library in an eukaryotic virus vector, comprising: 
 (a) providing host cells comprising a linear DNA virus genome which has been cleaved in a region non-essential for viral infectivity to produce a first viral fragment and a second viral fragment, wherein said first fragment is nonhomologous with said second fragment;    (b) providing a population of transfer plasmids comprising said insert polynucleotides in operable association with a vector transcriptional control region, a 5′ flanking region, and a 3′ flanking region; wherein said 5′ flanking region is homologous to said first viral fragment and said 3′ flanking region is homologous to said second viral fragment; and wherein said transfer plasmids are capable of homologous recombination with said first and second viral fragments such that a viable virus genome is formed;    (c) introducing said transfer plasmids into said host cells under conditions wherein a transfer plasmid and said first and second viral fragments undergo in vivo homologous recombination, thereby producing a viable modified virus genome comprising an insert polynucleotide; and    (d) collecting said modified virus genome.    
     
     
         139 . The method of  claim 138 , wherein said first and second viral fragments are produced by infecting said host cells with a virus comprising said linear DNA virus genome, and wherein said virus genome is cleaved in vivo.  
     
     
         140 . The method of  claim 138 , wherein said first and second viral fragments are produced by cleaving an isolated linear DNA virus genome in vitro, and wherein said first and second viral fragments are introduced into said host cells.  
     
     
         141 . The method of  claim 140 , wherein said virus genome comprises a first recognition site for a first restriction endonuclease; and wherein said first and second viral fragments are produced by digesting said viral genome with said first restriction endonuclease, and isolating said first and second viral fragments.  
     
     
         142 . The method of  claim 141 , wherein said virus genome further comprises a second recognition site for a second restriction endonuclease; and wherein said first and second viral fragments are produced by digesting said viral genome with said first restriction endonuclease and said second restriction endonuclease, and isolating said first and second viral fragments. claim  143 . The method of  claim 138 , wherein said modified virus genome is packaged in an infectious viral particle.  
     
     
         144 . The method of  claim 138 , wherein said linear DNA virus genome is a herpes virus genome.  
     
     
         145 . The method of  claim 138 , wherein said linear DNA virus genome is an adenovirus genome.  
     
     
         146 . The method of  claim 138 , wherein said linear DNA virus genome is a poxvirus genome.  
     
     
         147 . The method of  claim 146 , wherein said poxvirus genome is a vaccinia virus genome.  
     
     
         148 . The method of  claim 147 , wherein said vaccinia virus genome is a WR genome.  
     
     
         149 . The method of  claim 147 , wherein said vaccinia virus genome is a Modified Virus Ankara genome. claim  150 . The method of  claim 147 , wherein the molar ratio of said vaccinia virus genome to said transfer plasmid ranges from 1:1 to 1:10  
     
     
         151 . The method of claim  150 , wherein the molar ratio of said vaccinia virus genome to said transfer plasmid is 1:1  
     
     
         152 . The method of  claim 147 , wherein said non-essential region is within the Hind III F-fragment.  
     
     
         153 . The method of  claim 147 , wherein said non-essential region is within the fragment present in the L variant but deleted from the S variant of vaccinia virus.  
     
     
         154 . The method of  claim 146 , wherein said host cell further comprises a helper virus, and wherein said host cell is non-permissive for the production of infectious virus particles of said helper virus.  
     
     
         155 . The method of  claim 154 , wherein said helper virus is an avipoxvirus.  
     
     
         156 . The method of  claim 154 , wherein said helper virus is a fowlpox virus.  
     
     
         157 . The method of  claim 146 , wherein said transcriptional control region functions in the cytoplasm of a poxvirus-infected cell.  
     
     
         158 . The method of  claim 146 , wherein said transcriptional control region comprises a promoter.  
     
     
         159 . The method of  claim 158 , wherein said promoter is selected from the group consisting of a vaccinia virus p7.5 promoter, a synthetic early/late promoter, and a vaccinia virus MH5 early/late promoter.  
     
     
         160 . The method of  claim 158 , wherein said promoter is constitutive.  
     
     
         161 . The method of  claim 158 , wherein said promoter is a vaccinia virus p7.5 promoter.  
     
     
         162 . The method of  claim 159 , wherein said promoter is a synthetic early/late promoter.  
     
     
         163 . The method of  claim 159 , wherein said promoter is a vaccinia virus MH5 early/late promoter.  
     
     
         164 . The method of  claim 146 , wherein said transcriptional control region comprises a transcriptional termination region.