Modified piggyBac transposase polypeptide, polynucleotide encoding them, introducing carrier, kit, method of incorporating target sequence into cell genome, and method of producing cell
According to one embodiment, a modified piggyBac transposase polypeptide includes a piggyBac transposase amino acid sequence and a nuclear localization signal amino acid sequence.
1 . A polynucleotide encoding a modified piggyBac transposase, the polynucleotide comprising:
a) a base sequence encoding a piggyBac transposase comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1;
wherein the encoded amino acid sequence comprises:
a V at the position corresponding to amino acid 30 of SEQ ID NO: 1,
a P at the position corresponding to amino acid 103 of SEQ ID NO: 1,
an S at the position corresponding to amino acid 165 of SEQ ID NO: 1,
a V at the position corresponding to amino acid 282 of SEQ ID NO: 1,
a G at the position corresponding to amino acid 509 of SEQ ID NO: 1,
a K at the position corresponding to amino acid 538 of SEQ ID NO: 1, and
an S at the position corresponding to amino acid 571 of SEQ ID NO: 1;
and
b) a base sequence encoding a nuclear localization signal functionally linked to the encoded piggyBac transposase;
wherein the polynucleotide comprises the base sequence of SEQ ID NO: 17 or SEQ ID NO: 18, or the polynucleotide comprises the RNA equivalent of the base sequence of SEQ ID NO: 17 or SEQ ID NO: 18.
2 . The polynucleotide of claim 1 , wherein the polynucleotide is DNA or RNA.
3 . The polynucleotide of claim 1 wherein at least one codon among codons corresponding to amino acids constituting the modified piggyBac transposase is selected from the following codons for each of the amino acids:
GCU or GCC for alanine (A);
UGC or UGU for cysteine (C);
GAC or GAU for aspartic acid (D);
GAA or GAG for glutamic acid (E);
UUC or UUU for phenylalanine (F);
GGA or GGC for glycine (G);
CAC or CAU for histidine (H);
AUC or AUU for isoleucine (I);
AAA or AAG for lysine (K);
CUC or CUG for leucine (L);
AUG for methionine (M);
AAC or AAU for asparagine (N);
CCC or CCU for proline (P);
CAA or CAG for glutamine (Q);
AGA or AGG for arginine (R);
AGC, UCC or UCU for serine(S);
ACA or ACC for threonine (T);
GUC or GUG for valine (V);
UGG for tryptophan (W);
UAC or UAU for tyrosine (Y); and
UGA for a stop codon,
wherein A is adenine, U is uracil, C is cytosine, G is guanine, and when the polynucleotide is DNA, U is changed to thymine (T).
4 . The polynucleotide of claim 1 , wherein the polynucleotide is DNA and the base sequence encoding the nuclear localization signal comprises the sequence of SEQ ID NO: 8, 9 or 10.
5 . A polynucleotide encoding a modified piggyBac transposase, the polynucleotide comprising:
a) a base sequence encoding a piggyBac transposase comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1;
wherein the encoded amino acid sequence comprises:
a V at the position corresponding to amino acid 30 of SEQ ID NO: 1,
a P at the position corresponding to amino acid 103 of SEQ ID NO: 1,
an S at the position corresponding to amino acid 165 of SEQ ID NO: 1,
a V at the position corresponding to amino acid 282 of SEQ ID NO: 1,
a G at the position corresponding to amino acid 509 of SEQ ID NO: 1,
a K at the position corresponding to amino acid 538 of SEQ ID NO: 1, and
an S at the position corresponding to amino acid 571 of SEQ ID NO: 1;
b) a base sequence encoding a nuclear localization signal functionally linked to the encoded piggyBac transposase; and
c) a base sequence encoding a peptide that promotes cell division.
6 . A polynucleotide encoding a modified piggyBac transposase, the polynucleotide comprising:
a) a base sequence encoding a piggyBac transposase comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1;
wherein the encoded amino acid sequence comprises:
a V at the position corresponding to amino acid 30 of SEQ ID NO: 1,
a P at the position corresponding to amino acid 103 of SEQ ID NO: 1,
an S at the position corresponding to amino acid 165 of SEQ ID NO: 1,
a V at the position corresponding to amino acid 282 of SEQ ID NO: 1,
a G at the position corresponding to amino acid 509 of SEQ ID NO: 1,
a K at the position corresponding to amino acid 538 of SEQ ID NO: 1, and
an S at the position corresponding to amino acid 571 of SEQ ID NO: 1;
b) a base sequence encoding a nuclear localization signal functionally linked to the encoded piggyBac transposase; and
c) a base sequence encoding a peptide that promotes cell division;
wherein the polynucleotide comprises the base sequence of SEQ ID NO: 20.
7 . An introducing carrier for incorporating a target sequence into a cell genome comprising:
a lipid particle; and
the polynucleotide encoding a modified piggyBac transposase according to claim 1 ;
wherein the polynucleotide is encapsulated in the lipid particle.
8 . The introducing carrier of claim 1 ,
wherein the polynucleotide further comprises a base sequence encoding a peptide that promotes cell division.
9 . The introducing carrier of claim 8 , wherein donor DNA containing the target sequence is further encapsulated in the lipid particles.
10 . The introducing carrier of claim 7 , wherein the lipid particles contain, as a constituent component, a lipid compound represented by Formula (1-01) and/or a lipid compound represented by Formula (1-02).
11 . A kit for incorporating a target sequence into a cell genome, the kit comprising:
the polynucleotide encoding a modified piggyBac transposase of claim 1 ; and
a donor DNA containing a target sequence.
12 . The kit of claim 11 , wherein the polynucleotide and the donor DNA are encapsulated in lipid particle.
13 . A method of incorporating a target sequence into a cell genome, comprising introducing a donor DNA containing a target sequence into a cell with a polynucleotide encoding a modified piggyBac transposase according to claim 1 .
14 . The method of claim 13 ,
wherein the polynucleotide further comprises a base sequence encoding a peptide that promotes cell division.
15 . The method of claim 13 , wherein the introducing is performed using a liposome method, a lipofection method, an electroporation method, a sonoporation method, or a magnetofection method.
16 . The method of claim 15 ,
wherein the introducing is performed by using a liposome method comprising contacting the cell with lipid particles encapsulating the donor DNA, and the polynucleotide.
17 . A method of producing a cell, comprising introducing donor DNA containing a target sequence into a cell with a polynucleotide encoding a modified piggyBac transposase according to claim 1 .
18 . The method of claim 17 ,
wherein the polynucleotide further includes a base sequence encoding a peptide that promotes cell division.
19 . The method of claim 17 , wherein the introducing is performed using a liposome method, a lipofection method, an electroporation method, a sonoporation method, or a magnetofection method.
20 . The method of claim 19 ,
wherein the introducing is performed by a liposome method, and
lipid particles encapsulating the donor DNA, and the polynucleotide, are brought into contact with the cell.