Tol1 FACTOR TRANSPOSASE AND DNA INTRODUCTION SYSTEM USING THE SAME

Abstract

An object is to provide a Tol1 element transposase and a use thereof. Provided is a Tol1 element transposase containing (a) a protein having the amino acid sequence of SEQ ID No: 1 or (b) a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 1 and having an enzymatic activity for transferring Tol1 element. Further, provided is a polynucleotide encoding the transposase and an expression construct containing the polynucleotide therein. The present invention also provides a DNA introduction system including (a) a donor factor having such a structure that a desired DNA is inserted in a transposase gene-defected Tol1 element and (b) a helper factor containing the transposase or the polynucleotide.

1 - 34 . (canceled) 
     
     
         35 . A Tol1 element transposase comprising any of proteins selected from the group consisting of:
 (a) a protein having an amino acid sequence encoded by the base sequence of SEQ ID NO: 1; and   (b) a protein having the amino acid sequence of SEQ ID NO: 2.   
     
     
         36 . A DNA introduction system comprising:
 (a) a donor factor having such a structure that desired DNA is inserted in a transposase gene-defected Tol1 element; and   (b) a helper factor containing the transposase according to  claim 35 .   
     
     
         37 . The DNA introduction system according to  claim 36 , wherein the Tol1 element has the inverted repeat sequence of SEQ ID NO: 5 in the 5′ end region and the inverted repeat sequence of SEQ ID NO: 6 in the 3′ end region. 
     
     
         38 . The DNA introduction system according to  claim 36 , wherein the Tol1 element comprises DNA having the base sequence of any of SEQ ID NOs: 10 to 12. 
     
     
         39 . The DNA introduction system according to  claim 36 , wherein the Tol1 element comprises 5′ end side DNA and 3′ end side DNA obtained by deleting at least from the 158th base to the 1749th base counting from the 5′ end in the base sequence of SEQ ID NO: 10. 
     
     
         40 . The DNA introduction system according to  claim 36 , wherein the Tol1 element comprises DNA having the base sequence of SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO: 22. 
     
     
         41 . The DNA introduction system according to  claim 38 , wherein a target site duplicated sequence is connected to the 5′ end and the 3′ end of the toll element. 
     
     
         42 . The DNA introduction system according to  claim 41 , wherein the target site duplicated sequence comprises the sequence of any of SEQ ID NOs: 13 to 15. 
     
     
         43 . The DNA introduction system according to  claim 36 , wherein the desired DNA is a gene. 
     
     
         44 . The DNA introduction system according to  claim 36 , wherein the donor factor is a vector obtained by inserting a desired DNA in a transposase gene-defected Tol1 element, and the helper factor is a vector comprising a polynucleotide encoding a Tol1 element transposase, the polynucleotide comprising any of the nucleotide sequences selected from the group consisting of:
 (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; and   (b) the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.   
     
     
         45 . The DNA introduction system according to  claim 44 , wherein the vector being the helper factor further comprises a promoter operably linked to the polynucleotide. 
     
     
         46 . The DNA introduction system according to  claim 44 , wherein the vector being the helper factor further comprises a poly-A sequence connected to downstream of the polynucleotide. 
     
     
         47 . A DNA introduction method comprising a step of introducing the DNA introduction system according to  claim 36  to a target cell which is a vertebrate cell. 
     
     
         48 . The DNA introduction method according to  claim 47 , wherein the target cell is a vertebrate cell other than a cell in a state of a constituent factor of a human individual. 
     
     
         49 . The DNA introduction method according to  claim 47 , further comprising a step of introducing DNA different from the desired DNA introduced by the DNA introduction system to the target cell by utilizing Tol2 element. 
     
     
         50 . A method of transferring a specific DNA site on genomic DNA, comprising a step of supplying a transposase corresponding to Tol1 element or Tol2 element to a cell genetically manipulated with the DNA introduction method according to  claim 49 . 
     
     
         51 . A method of transferring a specific DNA site on genomic DNA, comprising a step of introducing the transposase according to  claim 35  into a cell having a transposase gene-defected Tol1 element on genomic DNA. 
     
     
         52 . The method according to  claim 51 , wherein another polynucleotide sequence is inserted in the Tol1 element. 
     
     
         53 . A cell genetically manipulated by the DNA introduction system according to  claim 36 . 
     
     
         54 . A DNA introducing kit, comprising:
 a donor factor comprising an expression construct containing a transposase gene-defected Tol1 element and having an insertion site; and   a helper factor comprising an expression construct containing the transposase according to  claim 35 .   
     
     
         55 . The DNA introducing kit according to  claim 54 , wherein the toll element comprises a structure having the insertion site between 5′ end side DNA and 3′ end side DNA, which is obtained by deleting at least from the 158th base to the 1749th base counting from the 5′ end in the base sequence of SEQ ID NO: 10. 
     
     
         56 . The DNA introducing kit according to  claim 54 , wherein the Tol1 element comprises a structure having the insertion site between DNA having the base sequence of SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO: 22. 
     
     
         57 . The DNA introducing kit according to  claim 54 , wherein the insertion site is made of a plurality of different kinds of restriction enzyme recognition sites.