US5612205A
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Homologous recombination in mammalian cells
Assignee: Pharming Intellectual Property BV
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Filing: 1991-08-28
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Granted: 1997-03-18
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Expires: 2011-08-28
Abstract
Abstract PCT No. PCT/US91/05917 Sec. 371 Date Mar. 1, 1993 Sec. 102(e) Date Mar. 1, 1993 PCT Filed Aug. 28, 1991 PCT Pub. No. WO92/03917 PCT Pub. Date Mar. 19, 1992The invention relates to methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and transgenic non-human mammals produced by such methods.
Inventors
- 👤 Robert M. Kay
- 👤 Anton Berns
- 👤 Paul Krimpenfort
- 👤 Frank Pieper
- 👤 Rein Strijker
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 23:58
CPC Classifications
A; A01; A01K; A01K67/00; A01K67/027; A01K67/0275; C; C07; C07K; C07K14/00; C07K14/435; C07K14/76; C07K14/765; C07K14/79; C07K16/00; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/87; C12N15/90; C12N15/902; C12N15/907; A01K2217/00; A01K2217/05; C12N2800/00; C12N2800/20; C12N2800/206; C12N2830/00; C12N2830/80; C12N2830/85
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
119 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragments homologously recombine in said cell to form a recombined DNA segment greater than 50 kb in length, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 2
Independent Claim
119 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragments homologously recombine in said cell to form a recombined DNA segment greater than 50 kb in length, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 3
Dependent Claim
12 words
The method of claim 1, wherein said cell is a non-human zygote.
Claim 4
Dependent Claim
13 words
The method of claim 1, wherein said co-introducing step is performed by microinjection.
Claim 5
Dependent Claim
13 words
The method of claim 1, wherein said recombined DNA segment encodes a protein.
Claim 6
Dependent Claim
12 words
The method of claim 4, wherein said protein is a human immunoglobulin.
Claim 7
Dependent Claim
11 words
The method of claim 4, wherein said protein is human lactoferrin.
Claim 8
Independent Claim
139 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb integrated into the genome of said cell, and said DNA segment does not encode a positive selection marker which can be used to detect homologous recombination between said first and said second fragments, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 9
Independent Claim
139 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb integrated into the genome of said cell, and said DNA segment does not encode a positive selection marker which can be used to detect homologous recombination between said first and said second fragments, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 10
Independent Claim
111 words
A method for generating a DNA segment inside an embryonic cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said embryonic cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said embryonic cell, wherein said mammalian embryonic cell is a non-human zygote or a mouse ES cell.
Claim 11
Independent Claim
111 words
A method for generating a DNA segment inside an embryonic cell comprising the step of introducing at least a first and a second DNA fragment different from the first DNA fragment into a recombination competent mammalian cell, said first DNA fragment having a sequence portion having at least 80% sequence identity to a sequence portion of said second DNA fragment, wherein said first and second DNA fragment homologously recombine in said embryonic cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said embryonic cell, wherein said mammalian embryonic cell is a non-human zygote or a mouse ES cell.
Claim 12
Dependent Claim
13 words
The method of claim 8, wherein said embryonic cell is a non-human zygote.
Claim 13
Independent Claim
139 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first, a second, and a third DNA fragment, each fragment different from the others, into a recombination competent mammalian cell, said second fragment having two sequence portions, one of which has at least 80% sequence identity to a sequence portion of the first fragment, the other of which has at least 80% sequence identity to a portion of the third fragment, wherein said first, second and third DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 14
Independent Claim
139 words
A method for generating a DNA segment inside a cell comprising the step of introducing at least a first, a second, and a third DNA fragment, each fragment different from the others, into a recombination competent mammalian cell, said second fragment having two sequence portions, one of which has at least 80% sequence identity to a sequence portion of the first fragment, the other of which has at least 80% sequence identity to a portion of the third fragment, wherein said first, second and third DNA fragment homologously recombine in said cell to form a recombined DNA segment of greater than 50 kb, said recombined DNA segment being integrated into the genome of said cell, wherein said cell is selected from the group consisting of endothelial cells, epithelial cells, myoblasts, hepatocytes, leukocytes, fibroblasts, mouse ES cells, and non-human zygotes.
Claim 15
Dependent Claim
12 words
The method of claim 10, wherein said cell is a non-human zygote.
Claim 16
Dependent Claim
13 words
The method of claim 10, wherein said introducing step is performed by microinjection.
Claim 17
Dependent Claim
13 words
The method of claim 10, wherein said recombined DNA segment encodes a protein.
Claim 18
Dependent Claim
10 words
The method of claim 1, wherein the cell is murine.
Claim 19
Dependent Claim
13 words
The method of claim 14, wherein the cell is an embryonic stem cell.
References Cited (Backward Citations)
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