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US5789215A
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Gene targeting in animal cells using isogenic DNA constructs
Assignee: Genpharm International Inc
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Filing: 1997-08-07
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Granted: 1998-08-04
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Expires: 2017-08-07
Abstract
Abstract The present invention provides novel methods for modifying the genome of an animal cell which typically comprise the steps of: constructing a DNA molecule in which desired sequence modifications are contained in a segment of DNA (a "targeting DNA") that is substantially isogenic with a DNA in the cell genome (a."target DNA"); introducing the targeting DNA construct into the cell (e.g., by microinjection, electroporation, transfection, or calcium phosphate precipitation); and selecting cells in which the desired sequence modifications have been introduced into the genome via homologous recombination.
Inventors
- 👤 Anton Berns
- 👤 Els Robanus Maandag
- 👤 Hein te Riele
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 16:48
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/87; C12N15/90; C12N15/902
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
157 words
A method for modifying the genome of a target cell of a multicellular animal by homologous recombination between a target DNA sequence in the target cell genome and a targeting DNA molecule introduced into the cell, said method comprising: a) constructing a targeting DNA molecule in which a desired sequence modification is contained in a targeting segment of DNA that is isogenic with the target DNA sequence in the target cell genome; b) introducing said targeting DNA molecule into target cells; and c) isolating a target cell in which said targeting DNA molecule is incorporated into its genome following homologous recombination between said targeting DNA molecule and said target DNA sequence thereby modifying the cell genome of said of said target cell of said multicellular animal; wherein the introducing step and the isolating step are performed in vitro, and further provided that where the target cell is an embryonic stem cell, the embryonic stem cell is murine.
Claim 2
Independent Claim
157 words
A method for modifying the genome of a target cell of a multicellular animal by homologous recombination between a target DNA sequence in the target cell genome and a targeting DNA molecule introduced into the cell, said method comprising: a) constructing a targeting DNA molecule in which a desired sequence modification is contained in a targeting segment of DNA that is isogenic with the target DNA sequence in the target cell genome; b) introducing said targeting DNA molecule into target cells; and c) isolating a target cell in which said targeting DNA molecule is incorporated into its genome following homologous recombination between said targeting DNA molecule and said target DNA sequence thereby modifying the cell genome of said of said target cell of said multicellular animal; wherein the introducing step and the isolating step are performed in vitro, and further provided that where the target cell is an embryonic stem cell, the embryonic stem cell is murine.
Claim 3
Dependent Claim
13 words
A method according to claim 1, wherein the multicellular animal is a mammal.
Claim 4
Dependent Claim
23 words
A method according to claim 1, wherein the desired sequence modification in the targeting DNA segment comprises an insertion of a selectable marker.
Claim 5
Dependent Claim
22 words
A method according to claim 3, wherein the selectable marker is a gene conferring resistance to a compound inhibitory to cell growth.
Claim 6
Dependent Claim
22 words
A method according to claim 3 wherein the selectable marker is a gene conferring the ability to grow on a selected substrate.
Claim 7
Dependent Claim
18 words
A method according to claim 1, wherein the targeting DNA molecule is introduced into the cell by microinjection.
Claim 8
Dependent Claim
14 words
A method according to claim 1, wherein the target DNA is an immunoglobulin gene.
Claim 9
Dependent Claim
23 words
The method of claim 1 further comprising isolating the targeting segment of DNA, and incorporating the isolated targeting segment into the targeting construct.
Claim 10
Dependent Claim
20 words
The method of claim 8, wherein the targeting segment and the target cell are from the same inbred mouse strain.
Claim 11
Dependent Claim
25 words
The method of claim 9, wherein the targeting segment is from a strain 129 mouse and the target cell is from a strain 129 mouse.
Claim 12
Dependent Claim
95 words
In a method of in vitro gene targeting comprising introducing a targeting DNA construct bearing a desired DNA sequence modification into a target cell of a multicellular eukaryotic organism in vitro whereby the construct undergoes homologous recombination with a target DNA sequence in the genome of the target cell thereby introducing the desired sequence modification into the genome of said cell, the improvement comprising preparing said targeting DNA construct by introducing said desired DNA sequence modification into a targeting segment of DNA that is isogenic with the target DNA sequence in the target cell genome.
Claim 13
Dependent Claim
95 words
In a method of in vitro gene targeting comprising introducing a targeting DNA construct bearing a desired DNA sequence modification into a target cell of a multicellular eukaryotic organism in vitro whereby the construct undergoes homologous recombination with a target DNA sequence in the genome of the target cell thereby introducing the desired sequence modification into the genome of said cell, the improvement comprising preparing said targeting DNA construct by introducing said desired DNA sequence modification into a targeting segment of DNA that is isogenic with the target DNA sequence in the target cell genome.
Claim 14
Dependent Claim
19 words
The method of claim 11 wherein the targeting segment of DNA is at least 1000 base pairs in length.
Claim 15
Dependent Claim
37 words
The method of claim 11 wherein the improvement further comprises the additional step of isolating said targeting segment of DNA is from a cell of the same multicellular eukaryotic organism from which the target cell is prepared.
Claim 16
Dependent Claim
14 words
The method of claim 11, wherein the multicellular eucaryotic organism is a noninbred animal.
Claim 17
Dependent Claim
13 words
The method of claim 11, wherein the multicellular euracyotic organism is a mammal.
Claim 18
Dependent Claim
23 words
The method of claim 11 further comprising isolating the targeting segment of DNA, and incorporating the isolated targeting segment into the targeting construct.
Claim 19
Dependent Claim
20 words
The method of claim 16 wherein the targeting segment and the target cell are from the same inbred mouse strain.
Claim 20
Dependent Claim
25 words
The method of claim 17, wherein the targeting segment is from a strain 129 mouse and the target cell is from a strain 129 mouse.
References Cited (Backward Citations)
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