Trap vectors and gene trapping using the same

Abstract

A trap vector containing a loxP sequence composed of inverted repeat sequence 1, a spacer sequence and inverted repeat sequence 2 in this order, the loxP sequence being a mutant loxP wherein a part of the inverted repeat sequence 1 or 2 is mutated.

1 . A trap vector comprising a loxP sequence and a lox 71  sequence,
 wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ;   wherein the lox 71  sequence comprises a nucleotide sequence shown in SEQ ID NO:15; and   wherein the trap vector is suitable for use in mammalian cells.   
     
     
         2 . A trap vector comprising a loxP sequence and a lox 66  sequence,
 wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ;   wherein the lox 66  comprises a nucleotide sequence shown in SEQ ID NO: 16; and   wherein the trap vector is suitable for use in mammalian cells.   
     
     
         3 . A vector generated from recombination between:
 (a) a trap vector comprising a loxP sequence and a mutant loxP sequence, wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ; wherein the mutant loxP sequence comprises a sequence of which a part of said inverted repeat sequence  1  of loxP is mutated; and   (b) a trap vector comprising a mutant loxP sequence, wherein the mutant loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ; wherein a part of said inverted repeat sequence  2  is mutated, or   (c) a trap vector comprising a loxP sequence and a mutant loxP sequence, wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence and inverted repeat sequence  2 ; wherein the mutant loxP sequence comprises a sequence of which a part of said inverted repeat sequence  2  of loxP is mutated; and   (d) a trap vector comprising a mutant loxP sequence, wherein the mutant loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence and inverted repeat sequence  2 ; wherein a part of said inverted repeat sequence  1  is mutated,   wherein recombination of the mutant loxP occurs more efficiently than the reverse reaction as compared to wild-type loxP.   
     
     
         4 . The vector of  claim 3 , wherein said vector does not undergo recombination with another loxP. 
     
     
         5 . A method of gene trapping, comprising the steps of:
 introducing the trap vector of  claim 1  or  2  into embryonic stem cells;   culturing the embryonic stem cells;   selecting those cells which exhibit a pattern of single copy integration of the trap vector; and   isolating the trapped gene.   
     
     
         6 . Embryonic stern cells into which the trap vector of  claim 1  or  2  is introduced. 
     
     
         7 . A method of gene trapping, said method comprising the steps of: introducing into embryonic stem cells:
 (a) a trap vector comprising a loxP sequence, a marker gene and a mutant loxP sequence, wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ; wherein the mutant loxP sequence comprises a sequence of which a part of said inverted repeat sequence  1  of loxP is mutated, and   (b) a trap vector comprising a mutant loxP sequence, wherein the mutant loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence, and inverted repeat sequence  2 ; wherein a part of said inverted repeat sequence  2  is mutated, or   (c) a trap vector comprising a loxP sequence, a marker gene and a mutant loxP sequence, wherein the loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence and inverted repeat sequence  2 ; wherein the mutant loxP sequence comprises a sequence of which a part of said inverted repeat sequence  2  of loxP is mutated; and   (d) a trap vector comprising a mutant loxP sequence, wherein the mutant loxP sequence comprises in sequential order inverted repeat sequence  1 , a spacer sequence and inverted repeat sequence  2 ; wherein a part of said inverted repeat sequence  1  is mutated,   wherein recombination of the mutant loxP occurs more efficiently than the reverse reaction as compared to wild-type loxP, and   wherein trap vector (a) or (b) further comprises at least one splice acceptor site and at least one internal ribosomal entry site;   culturing the embryonic stem cells;   selecting those cells which exhibit a pattern of single copy integration of the trap vector; and   isolating the trapped gene.   
     
     
         8 . The method according to  claim 7 , wherein the trap vector further comprises pA and pV, wherein pA is located downstream of the marker gene.