Trichoplusia ni piggyBac transposases with reduced integration activity
The present invention is directed to nucleic acid and amino acid sequences of a novel piggyBac transposase enzymes created by modifying the transposase of Trichoplusia ni . The piggyBac transposases of the present invention are functionally active or hyperactive for excision and have decreased integration activity compared to wild type Trichoplusia ni piggyBac transposase enzyme. These transposases are ideal for use in methods of transforming cells and organisms. In particular embodiments, the present invention provides methods of transient integration and expression of transgenes.
1. An improved Trichoplusia ni piggyBac transposase comprising a substitution at one or more of amino acid positions 245, 275, 277, 341, 372, 375, and 388 of SEQ ID NO: 52, wherein SEQ ID NO: 52 consists of amino acid residues 181-540 of the wild type Trichoplusia ni piggyBac transposase, and wherein the improved transposase exhibits enhanced piggyBac transposon excision activity and no or significantly diminished piggyBac transposon integration as compared to a wild type Trichoplusia ni piggyBac transposase.
2. The improved transposase according to claim 1 , wherein an arginine at one or more of amino acid positions 245, 275, 277, 341, 372 and 388 is replaced with an alanine.
3. The improved transposase according to claim 1 wherein a lysine at amino acid position 375 is replaced with an alanine.
4. The improved transposase according to claim 1 , wherein an arginine at amino acid position 275 is replaced with alanine and an arginine at amino acid position 277 is replaced with alanine.
5. The improved transposase according to claim 1 , wherein an arginine at amino acid position 372 is replaced with alanine and a lysine at amino acid position 375 is replaced with alanine.
6. The improved transposase according to claim 1 , wherein an arginine at amino acid position 388 is replaced with alanine.
7. A method of reversibly transforming a cell comprising the steps of:
using a piggyBac transposon to insert a nucleic acid sequence into the genome of the cell thereby transforming the cell; and
exposing the cell to the improved transposase of any one of claims 1 , 2 - 3 , and 4 - 6 , thereby removing the nucleic acid sequence.