Recombinase-Based Methods for Producing Expression Vectors and Compositions for Use in Practicing the Same

Abstract

Methods are provided for producing a vector that includes at least one splicable intron. In the subject methods, intron containing vectors are produced from donor and acceptor vectors that each include a site specific recombinase site, where the subject donor and acceptor vectors further include splice donor and acceptor sites that, upon site specific recombination of the donor and acceptor vectors, define an intron in the product vector of the recombination step. Also provided are compositions for use in practicing the subject methods, including the donor and acceptor vectors themselves, as well as systems and kits that include the same. The subject invention finds use in a variety of different applications, including the production of expression vectors that encode C-terminal tagged fusion proteins, the production of expression vectors that encode pure protein and not a fusion thereof, and the like.

1 . A sequence specific recombinase based system for use in preparing an intron containing vector, said system comprising:
 a donor vector comprising at least one splice site and an acceptor vector comprising at least one splice site, wherein said donor and acceptor vectors each comprise at least one recombinase recognition site.   
     
     
         2 . The system according to  claim 1 , wherein one of said donor and acceptor vectors comprises two recombinase recognition sites and the other of said donor and acceptor vectors comprises a single recombinase recognition site, wherein all of said recombinase recognition sites are able to recombine with each other. 
     
     
         3 . The system according to  claim 2 , wherein said donor vector comprises two recombinase recognition sites and said acceptor vector comprises a single recombinase recognition site. 
     
     
         4 . The system according to  claim 3 , wherein said two recombinase recognition sites on said donor vector are oriented in the same direction. 
     
     
         5 . The system according to  claim 2 , wherein said donor vector comprises a single recombinase recognition site and said acceptor comprises two recombinase recognition sites. 
     
     
         6 . The system according to  claim 5 , wherein said two recombinase recognition sites of said acceptor vector are oriented in the same direction. 
     
     
         7 . The system according to  claim 1 , wherein said system further comprises a sequence specific recombinase. 
     
     
         8 . The system according to  claim 1 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, aft sites, dif sites and frt sites. 
     
     
         9 . The system according to  claim 1 , wherein said donor and acceptor vectors are plasmids, cosmids, bacs, yacs or viruses. 
     
     
         10 . The system according to  claim 1 , wherein said system further comprises a host cell. 
     
     
         11 . The system according to  claim 1 , wherein each of said donor and acceptor vectors comprise a splice donor and a splice acceptor sequence. 
     
     
         12 . A donor vector comprising:
 (a) at least one recombinase recognition site; and   (b) a splice sequence.   
     
     
         13 . The donor vector according to  claim 12 , wherein said donor vector comprises first and second recombinase recognition sites oriented in the same direction and flanking a portion of a selectable marker, wherein said first and second recombinase recognition sites are able to recombine with each other. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . An acceptor vector comprising:
 (a) at least one recombinase recognition site; and   (b) a splice sequence.   
     
     
         17 . The acceptor vector according to  claim 16 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites. 
     
     
         18 - 23 . (canceled) 
     
     
         24 . A method of producing an intron containing vector, said method comprising: combining a splice sequence comprising donor vector and a splice sequence comprising acceptor vector with a recombinase under conditions sufficient for site-specific recombination to occur to produce said intron containing vector. 
     
     
         25 . The method according to  claim 24 , wherein said donor vector comprises two recombinase recognition sites and said acceptor vector comprises a single recombinase recognition site. 
     
     
         26 . The method according to  claim 24 , wherein said donor vector comprises a single recombinase recognition site and said acceptor vector comprises two recombinase recognition sites. 
     
     
         27 . The method according to  claim 24 , wherein said sequence specific recombinase is selected from the group consisting of: recombinases, transposases and integrases. 
     
     
         28 . The method according to  claim 24 , wherein said sequence specific recombinase is Cre recombinase. 
     
     
         29 . The method according to  claim 24 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites. 
     
     
         30 . (canceled) 
     
     
         31 . An intron containing vector comprising:
 (a) at least one recombinase recognition site; and   (b) a spliceable intron.   
     
     
         32 . The vector according to  claim 31 , wherein said vector comprises first and second recombinase recognition sites oriented in the same direction. 
     
     
         33 . The vector according to  claim 32 , wherein said vector further comprises:
 an expression cassette for a protein of interest divided into two subparts that flank said first recombinase recognition; and a functional marker divided into two sub-parts that flank said second recombinase recognition site.   
     
     
         34 . The vector according to  claim 31 , wherein said recombinase recognition sites are selected from the group consisting of: lox sites, att sites, dif sites and frt sites. 
     
     
         35 - 37 . (canceled)