Gene targeting in animal cells using isogenic DNA constructs
DCFirst Claim
1. 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.
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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.
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Citations
18 Claims
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1. 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:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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- 11. 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.
Specification