Modified integrase and methods of use
The invention includes modified enzymes such as integrases which maintain all or part of their enzymatic activity and can localize to the nucleus.
1 . An enzyme which facilitates insertion of a nucleotide sequence into the genome of a cell wherein the enzyme has been modified to contain an NLS.
2 . The enzyme of claim 1 wherein the enzyme is an integrase.
3 . The enzyme of claim 1 wherein the integrase contains the NLS at a position that is not conserved among serine integrases.
4 . The enzyme of claim 1 wherein the enzyme is present in a reproductive cell.
5 . The enzyme of claim 1 wherein the enzyme is present in a germinal disc.
6 . The enzyme of claim 5 wherein the germinal disc is an embryo.
7 . A serine integrase containing an NLS.
8 . A phiC31 integrase containing an NLS.
9 . A mutant integrase wherein an NLS is contained in the integrase amino acid sequence at a position that is nonconserved.
10 . The integrase of claim 9 wherein the integrase is a tyrosine recombinase.
11 . The integrase of claim 9 wherein the integrase is a serine recombinase.
12 . A phiC31 integrase wherein the NLS is inserted between amino acid 228 and amino acid 242.
13 . A cell containing an integrase which contains an NLS wherein the cell contains a nucleotide sequence comprising a heterologous coding sequence.
14 . The cell of claim 13 wherein the heterologous coding sequence encodes a pharmaceutical protein.
15 . The cell of claim 13 wherein the integrase is encoded by RNA present in the cell.
16 . The cell of claim 13 wherein the integrase is encoded by DNA present in the cell.
17 . The cell of claim 13 wherein the cell contains a heterologous integration site.
18 . The cell of claim 17 wherein the integration site is a serine recombinase recombination site.
19 . The cell of claim 17 wherein the integration site is an attP site.
20 . The cell of claim 17 wherein the integration site is an attB site.
21 . A method comprising introducing an integrase-NLS fusion protein and nucleic acid which comprises an integration sequence into a cell wherein the cell contains an integration site in its genome.
22 . The method of claim 21 wherein the cell contains an increased volume of cytoplasm.
23 . The method of claim 22 wherein the increase volume of cytoplasm is relative to the volume of cytoplasm contained in an avian fibroblast cell in culture.
24 . The method of claim 21 wherein the NLS is contained in the integrase.
25 . The method of claim 21 wherein the nucleic acid encodes a heterologous protein.
26 . The method of claim 25 wherein the heterologous protein is a pharmaceutical protein.
27 . The method of claim 21 wherein the nucleic acid is associated with a cationic polymer.
28 . The method of claim 21 wherein the integration site in the cell genome is heterologous.
29 . The method of claim 21 wherein the cell is resistant to the introduction of integrase protein.
30 . The method of claim 28 wherein the cell is an avian cell.
31 . The method of claim 21 wherein the cell is a cell of a germinal disc.
32 . The method of claim 21 wherein the cell is a cell of an avian embryo.
33 . The method of claim 21 wherein the integrase-NLS fusion protein is introduced by injection.