Gene targeting in animal cells using isogenic DNA constructs
First Claim
1. A method for modifying a preselected DNA sequence in a cell of a non-inbred animal by homologous recombination between a native target DNA sequence in the preselected DNA sequence and a targeting DNA sequence introduced into the cell, said method comprising:
- isolating cells in which preselected sequence modifications have been incorporated into the genome by homologous recombination between the target DNA and the targeting DNA, wherein the target DNA and the targeting DNA are substantially isogenic except for the preselected sequence modifications.
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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.
33 Citations
36 Claims
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1. A method for modifying a preselected DNA sequence in a cell of a non-inbred animal by homologous recombination between a native target DNA sequence in the preselected DNA sequence and a targeting DNA sequence introduced into the cell, said method comprising:
isolating cells in which preselected sequence modifications have been incorporated into the genome by homologous recombination between the target DNA and the targeting DNA, wherein the target DNA and the targeting DNA are substantially isogenic except for the preselected sequence modifications. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 18, 19, 20)
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9. A method for modifying a non-murine animal cell genome by homologous recombination between a target DNA sequence in the animal cell genome and a targeting DNA sequence introduced into the animal cell, said method comprising:
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introducing into cells to be targeted a DNA delivery molecule comprising the targeting DNA; and
isolating cells in which preselected sequence modifications have been incorporated into the genome by homologous recombination between the target DNA and the targeting DNA, wherein the target DNA and the targeting DNA are substantially isogenic except for the preselected sequence modifications. - View Dependent Claims (10, 11, 12, 13, 14)
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15. A method for modifying a cell genome of an animal by homologous recombination between a target DNA sequence in the animal cell genome and a targeting DNA sequence introduced into the animal cell, said method comprising:
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introducing a DNA delivery molecule comprising the targeting DNA into cells to be targeted, wherein the targeting DNA was prepared from cells of the same individual animal or a sibling thereof; and
isolating cells in which preselected sequence modifications have been incorporated into the genome by homologous recombination between the target DNA and the targeting DNA, wherein the target DNA and the targeting DNA are substantially isogenic except for the preselected sequence modifications.
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16. A method for enhancing homologous recombination between a native target DNA sequence in a non-murine mammalian cell line and a targeting DNA sequence introduced into the cell line, said method comprising the steps of:
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isolating targeting DNA derived from a second cell line wherein said targeting DNA is substantially isogenic with the target DNA; and
introducing desired sequence-modifications into the targeting DNA; and
introducing a DNA delivery molecule comprising the targeting DNA into cells to be targeted; and
isolating cells in which one or more of the sequence modifications are incorporated into the genome by homologous recombination between the target DNA and the targeting DNA. - View Dependent Claims (17)
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21. A method for gene therapy of an animal comprising:
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introducing into cells of a first animal to be targeted a DNA delivery molecule comprising a targeting DNA sequence from a second animal, which sequence is capable of effecting homologous recombination with a substantially isogenic target DNA sequence, other than preselected sequence modifications, in the first animal cell genome;
isolating cells in which the preselected sequence modifications have been incorporated into the genome; and
introducing the modified cells into the first animal. - View Dependent Claims (22, 23, 24, 25, 26, 27)
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28. A composition comprising a collection of cells between about 10 to 90% of which exhibit a recombination event at a preselected native target DNA segment of the cells, which recombination event is selected from the group consisting of an addition of an exogenous DNA segment to the native DNA segment, a substitution of an exogenous DNA segment for the native DNA segment, and a deletion of the native DNA segment from the cell;
- wherein genomes of the cells exhibiting the event consist essentially of substantially isogenic DNA proximate to the recombination event except for the exogenous DNA.
- View Dependent Claims (29, 30, 31)
- 32. A non-human animal comprising cells with a homologous recombination event at a preselected native target DNA segment in the cell genome, wherein genomes of the cells consist essentially of substantially isogenic DNA proximate to the target DNA segment except for preselected sequence modifications which are incapable of undergoing homologous recombination in the cells unless linked to a second DNA segment homologous to the native target DNA segment.
Specification