Site specific recombination in avians
The invention includes transgenic avians and transgenic avian cells and methods for site specifically introducing nucleotide sequences into the genome of avians and avian cells.
1 . A method of producing a transgenic avian comprising:
introducing into an embryo of an avian a recombination site such that the recombination site is present in ovum of a mature avian developed from the embryo wherein the embryo does not normally comprise the recombination site,
introducing into the embryo of an avian or fertilized ovum of the mature avian, a nucleotide sequence comprising a second recombination site and a coding sequence;
introducing into the embryo of an avian or fertilized ovum of the mature avian a substance which facilitates insertion of the nucleotide sequence comprising a second recombination site and a coding sequence proximal to the recombination site; and
exposing the embryo of an avian or fertilized ovum of the mature avian to conditions which lead to development of a viable transgenic avian,
thereby producing a transgenic avian.
2 . The method of claim 1 wherein the avian selected from the group consisting of chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary.
3 . The method of claim 1 wherein the avian is a chicken.
4 . The method of claim 1 wherein the embryo or fertilized ovum is selected from the group consisting of a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stage XII embryo.
5 . The method of claim 1 wherein at least one of the recombination site or the nucleotide sequence comprising a second recombination site and a coding sequence is introduced into the embryo or fertilized ovum by a method selected from the group consisting of transfection, microinjection, cell fusion and lipofection.
6 . The method of claim 1 wherein the recombination site or nucleotide sequence comprising a second recombination site and a coding sequence is introduced into the embryo or fertilized ovum in the presence of PEI.
7 . The method of claim 1 wherein at least one of the recombination site or the second recombination site is isolated from a bacteriophage.
8 . The method of claim 1 wherein the recombination site is an attP site or an attB site.
9 . The method of claim 1 wherein the second recombination site is an attP site or an attB site.
10 . The method of claim 1 wherein the nucleotide sequence is stably incorporated into the genome of the embryo or fertilized ovum.
11 . The method of claim 1 wherein the substance is an enzyme.
12 . The method of claim 11 wherein the enzyme is a site specific recombinase.
13 . The method of claim 11 wherein the enzyme is selected from the group consisting of a serine recombinase and a tyrosine recombinase.
14 . The method of claim 11 wherein the enzyme is selected from the group consisting of ΦC31, TP901-1 and R4 serine recombinases.
15 . The method of claim 11 wherein the enzyme is ΦC31 serine recombinase.
16 . The method of claim 1 wherein the substance is nucleic acid.
17 . The method of claim 16 wherein the nucleic acid is DNA or RNA.
18 . The method of claim 17 wherein a vector comprises the DNA.
19 . The method of claim 17 wherein the nucleic acid encodes an enzyme.
20 . The method of claim 17 wherein the nucleic acid encodes a site specific recombinase.
21 . The method of claim 17 wherein the nucleic acid encodes an enzyme selected from the group consisting of a serine recombinase and a tyrosine recombinase.
22 . The method of claim 17 wherein the nucleic acid encodes a serine recombinase selected from the group consisting of ΦC31, TP901-1 and R4 serine recombinases.
23 . The method of claim 17 wherein the nucleic acid encodes a ΦC31 serine recombinase.
24 . The method of claim 1 wherein the nucleotide sequence comprises an expression cassette.
25 . The method of claim 1 wherein the coding sequence encodes a therapeutic composition.
26 . The method of claim 25 wherein the therapeutic composition comprises at least one of a light chain or a heavy chain of an antibody.
27 . The method of claim 25 wherein the therapeutic composition is a cytokine.
28 . The method of claim 25 wherein the therapeutic composition is selected from the group consisting of interferon, erythropoietin, and granulocyte-colony stimulating factor.
29 . The method of claim 1 wherein the coding sequence encodes a polypeptide present in an egg produced by the transgenic avian.
30 . An egg of claim 29 .
31 . The method of claim 1 wherein the recombination site is introduced into the embryo of an avian or fertilized ovum before fertilization.
32 . An ovum or sperm produced by a transgenic avian of claim 1 .
33 . A transgenic avian produced according to the method of claim 1 .
34 . A descendent of the transgenic avian of claim 1 .
35 . A method of producing a transgenic avian comprising:
introducing into an embryo of an avian a recombination site such that the recombination site is present in sperm of a mature avian developed from the embryo wherein the embryo does not normally comprise the recombination site,
fertilizing an ovum with sperm comprising the recombination site;
introducing into the ovum a nucleotide sequence comprising a second recombination site and a coding sequence and a substance which facilitates insertion of the nucleotide sequence comprising a second recombination site and coding sequence proximal to the recombination site; and
exposing the fertilized ovum to conditions which lead to development of a viable transgenic avian,
thereby producing a transgenic avian.
36 . The method of claim 35 wherein the avian is selected from the group consisting of chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary.
37 . The method of claim 35 wherein the avian is a chicken.
38 . The method of claim 35 wherein the embryo is selected from the group consisting of a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stage XII embryo.
39 . The method of claim 35 wherein at least one of the recombination site or the nucleotide sequence comprising a second recombination site and a coding sequence is introduced into the embryo or fertilized ovum by a method selected from the group consisting of transfection, microinjection, cell fusion and lipofection.
40 . The method of claim 35 wherein the recombination site or nucleotide sequence comprising a second recombination site and a coding sequence is introduced in the presence of PEI.
41 . The method of claim 35 wherein at least one of the recombination site or the second recombination site is isolated from a bacteriophage.
42 . The method of claim 35 wherein the recombination site is an attP site or an attB site.
43 . The method of claim 35 wherein the second recombination site is an attP site or an attB site.
44 . The method of claim 35 wherein the nucleotide sequence is stably incorporated into the genome of the embryo or fertilized ovum.
45 . The method of claim 35 wherein the substance is an enzyme.
46 . The method of claim 45 wherein the enzyme is a site specific recombinase.
47 . The method of claim 45 wherein the enzyme is selected from the group consisting of a serine recombinase and a tyrosine recombinase.
48 . The method of claim 45 wherein the enzyme is selected from the group consisting of ΦC31, TP901-1 and R4 serine recombinases.
49 . The method of claim 45 wherein the enzyme is ΦC31 serine recombinase.
50 . The method of claim 35 wherein the substance is nucleic acid.
51 . The method of claim 50 wherein the nucleic acid is DNA or RNA.
52 . The method of claim 51 wherein a vector comprises the DNA.
53 . The method of claim 50 wherein the nucleic acid encodes an enzyme.
54 . The method of claim 50 wherein the nucleic acid encodes a site specific recombinase.
55 . The method of claim 50 wherein the nucleic acid encodes an enzyme selected from the group consisting of a serine recombinase and a tyrosine recombinase.
56 . The method of claim 50 wherein the nucleic acid encodes a serine recombinase selected from the group consisting of ΦC31, TP901-1 and R4 serine recombinases.
57 . The method of claim 50 wherein the nucleic acid encodes a ΦC31 serine recombinase.
58 . The method of claim 35 wherein the nucleotide sequence comprises an expression cassette.
59 . The method of claim 35 wherein the coding sequence encodes a therapeutic composition.
60 . The method of claim 59 wherein the therapeutic composition comprises at least one of a light chain or a heavy chain of an antibody.
61 . The method of claim 60 wherein the antibody is a human antibody.
62 . The method of claim 59 wherein the therapeutic composition is a cytokine.
63 . The method of claim 59 wherein the therapeutic composition is selected from the group consisting of interferon, erythropoietin, and granulocyte-colony stimulating factor.
64 . The method of claim 35 wherein the coding sequence encodes a polypeptide present in an egg produced by the transgenic avian.
65 . An egg of claim 64 .
66 . The method of claim 35 wherein the recombination site is introduced into the embryo of an avian or fertilized ovum before fertilization.
67 . An ovum or sperm produced by a transgenic avian of claim 35 .
68 . A transgenic avian produced according to the method of claim 35 .
69 . A descendent of the transgenic avian of claim 35 .
70 . A method of modifying an avian cell genome comprising:
introducing into an avian cell a recombination site such that the recombination site is inserted into the avian cell genome wherein the genome does not normally comprise the recombination site;
introducing a nucleotide sequence comprising a second recombination site and a coding sequence into the avian cell or progeny cell thereof; and
introducing into the avian cell or progeny cell thereof a substance which facilitates insertion of the nucleotide sequence comprising a second recombination site and a coding sequence proximal to the recombination site,
thereby modifying an avian cell genome.
71 . The method of claim 70 wherein the avian cell is a cell of an avian.
72 . The method of claim 70 wherein the avian cell is a chicken cell.
73 . The method of claim 70 wherein the avian cell is a germ line cell.
74 . The method of claim 70 wherein the avian cell is an ovum or an embryo.
75 . The method of claim 70 wherein the avian cell an embryo cell and the embryo is selected from the group consisting of a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stage XII embryo.
76 . The method of claim 70 wherein at least one of the recombination site or the nucleotide sequence comprising a second recombination site and a coding sequence is introduced into the avian cell genome by a method selected from the group consisting of cell fusion, lipofection, transfection and microinjection.
77 . The method of claim 70 wherein the recombination site or nucleotide sequence comprising a second recombination site and a coding sequence is introduced into the avian cell genome in the presence of PEI.
78 . The method of claim 70 wherein at least one of the recombination site or the second recombination site is isolated from a bacteriophage.
79 . The method of claim 70 wherein the recombination site is an attP site or an attB site.
80 . The method of claim 70 wherein the second recombination site is an attP site or an attB site.
81 . The method of claim 70 wherein the nucleotide sequence is stably incorporated into the genome of the avian cell.
82 . The method of claim 70 wherein the substance is an enzyme.
83 . The method of claim 82 wherein the enzyme is a site specific recombinase.
84 . The method of claim 82 wherein the enzyme is selected from the group consisting of a serine recombinase and a tyrosine recombinase.
85 . The method of claim 82 wherein the enzyme is ΦC31 serine recombinase.
86 . The method of claim 70 wherein the substance is nucleic acid.
87 . The method of claim 86 wherein the nucleic acid encodes an enzyme.
88 . The method of claim 86 wherein the nucleic acid encodes a site specific recombinase.
89 . The method of claim 86 wherein the nucleic acid encodes an enzyme selected from the group consisting of a serine recombinase and a tyrosine recombinase.
90 . The method of claim 82 wherein the nucleic acid encodes a ΦC31 serine recombinase.
91 . The method of claim 70 wherein the coding sequence encodes a therapeutic composition.
92 . The method of claim 91 wherein the therapeutic composition comprises at least one of a light chain or a heavy chain of an antibody.
93 . The method of claim 91 wherein the therapeutic composition is a cytokine.
94 . The method of claim 91 wherein the therapeutic composition is selected from the group consisting of interferon, erythropoietin, and granulocyte-colony stimulating factor.
95 . A cell produced according to the method of claim 70 .
96 . A transgenic avian comprising a recombination site present in the genome of a cell wherein the genome does not normally comprise the recombination site.