TRANSGENIC BIRD PRODUCING ERYTHROPOIETIN AND METHOD OF CONSTRUCTING THE SAME
The present invention has its object to provide a transgenic bird producing erythropoietin at high concentration levels as well as a method for constructing the same. The present invention provides a G0 transgenic chimera bird as obtained by incubating a fertilized avian egg, infecting the early embryo formed after egg laying, except for the blastoderm stage immediately following egg laying, with a replication-deficient retroviral vector containing a foreign erythropoietin gene and allowing the embryo to hatch.
1 . A G0 transgenic chimera bird as obtained by incubating a fertilized avian egg, infecting the early embryo formed after egg laying, except for the blastoderm stage immediately following egg laying, with a replication-deficient retroviral vector containing a foreign erythropoietin gene and allowing the embryo to hatch.
2 . The G0 transgenic chimera bird according to claim 1 ,
wherein the foreign erythropoietin gene is a mammal-derived one.
3 . The G0 transgenic chimera bird according to claim 2 ,
wherein the foreign erythropoietin gene is a human-derived one.
4 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the foreign erythropoietin gene is under the control of a non-tissue-specific promoter.
5 . The G0 transgenic chimera bird according to claim 4 ,
wherein the non-tissue-specific promoter is a chicken β-actin gene-derived one.
6 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the foreign erythropoietin gene is under the control of a tissue-specific promoter.
7 . The G0 transgenic chimera bird according to claim 6 ,
wherein the tissue-specific promoter is a chicken ovalbumin gene-derived one.
8 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the foreign erythropoietin gene has a posttranscriptional regulatory element added thereto.
9 . The G0 transgenic chimera bird according to claim 8 ,
wherein the posttranscriptional regulatory element is WPRE.
10 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the replication-deficient retroviral vector is a MoMLV- or MSCV-derived one.
11 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the replication-deficient retroviral vector is of the VSV-G pseudotype.
12 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the infection is carried out using, as the replication-deficient retroviral vector, a virus-containing solution having a replication-deficient retroviral vector titer of not lower than 1×10 8 cfu/ml but not higher than 1×10 10 cfu/ml.
13 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the early embryo is one formed not earlier by 24 hours but not later by 60 hours than the start of incubation.
14 . The G0 transgenic chimera bird according to claim 13 ,
wherein the early embryo is one formed not earlier by 48 hours but not later by 60 hours than the start of incubation.
15 . The G0 transgenic chimera bird according to any one of claim 1 ,
wherein the infection is carried out by microinjection into the heart or a blood vessel formed in the early embryo.
16 . The G0 transgenic chimera bird according to claim 15 ,
wherein the infection is carried out by microinjection into the heart formed in the early embryo.
17 . A G1 transgenic bird and a descendant thereof as obtained by mating the G0 transgenic chimera bird according to any one of claim 1 with another G0 transgenic chimera bird of the same kind, a descendant thereof or a wild type bird.
18 . A G2 transgenic bird and a descendant thereof as obtained by mating the G1 transgenic bird according to any of claim 17 with another G1 transgenic bird of the same kind, a descendant thereof, a wild type bird or a G0 transgenic chimera bird obtained by incubating a fertilized avian egg, infecting embryo formed after egg laying, except for the blastoderm stage immediately following egg laying, with a replication-deficient retroviral vector containing a foreign erythropoietin gene and allowing the embryo to hatch.
19 . The transgenic bird according to claim 1
which is a chicken or quail.
20 . An egg laid by the transgenic bird according to any one of claim 1 .
21 . A method for producing erythropoietin
which comprises extracting and purifying erythropoietin from the egg according to claim 20 .
22 . A method for producing erythropoietin
which comprises extracting and purifying erythropoietin from the blood of the transgenic bird according to claim 1 .
23 . A sperm of the transgenic bird according to claim 1 .
24 . A method for constructing a G0 transgenic chimera bird
which comprises incubating a fertilized avian egg, infecting the early embryo formed after egg laying, except for the blastoderm stage immediately following egg laying, with a replication-deficient retroviral vector containing a foreign erythropoietin gene and allowing the embryo to hatch.
25 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the foreign erythropoietin gene is a mammal-derived one.
26 . The method for constructing a G0 transgenic chimera bird according to claim 25 ,
wherein the foreign erythropoietin gene is a human-derived one.
27 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the foreign erythropoietin gene is under the control of a non-tissue-specific promoter.
28 . The method for constructing a G0 transgenic chimera bird according to claim 27 ,
wherein the non-tissue-specific promoter is a chicken β-actin gene-derived one.
29 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the foreign erythropoietin gene is under the control of a tissue-specific promoter.
30 . The method for constructing a G0 transgenic chimera bird according to claim 29 ,
wherein the tissue-specific promoter is a chicken ovalbumin gene-derived one.
31 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the foreign erythropoietin gene has a posttranscriptional regulatory element added thereto.
32 . The method for constructing a G0 transgenic chimera bird according to claim 31 ,
wherein the posttranscriptional regulatory element is WPRE.
33 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the replication-deficient retroviral vector is a MoMLV- or MSCV-derived one.
34 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the replication-deficient retroviral vector is of the VSV-G pseudotype.
35 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the infection is carried out using, as the replication-deficient retroviral vector, a virus-containing solution having a replication-deficient retroviral vector titer of not lower than 1×10 8 cf/ml but not higher than 1×10 10 cfu/ml.
36 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the early embryo is one formed not earlier by 24 hours but not later by 60 hours than the start of incubation.
37 . The method for constructing a G0 transgenic chimera bird according to claim 36 ,
wherein the early embryo is one formed not earlier by 48 hours but not later by 60 hours than the start of incubation.
38 . The method for constructing a G0 transgenic chimera bird according to claim 24 ,
wherein the infection is carried out by microinjection into the heart or a blood vessel formed in the early embryo.
39 . The method for constructing a G0 transgenic chimera bird according to claim 38 ,
wherein the infection is carried out by microinjection into the heart formed in the early embryo.