Method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same
First Claim
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1. A method for inserting a desired DNA at a target site in the genome of a mammalian cell which comprises the following steps:
- (i) transfecting or transforming a mammalian cell in vitro with a marker plasmid comprising the following sequences;
(a) a first DNA fragment which comprises a region that is heterologous to the mammalian cell genome and provides a unique site for homologous recombination when it is integrated in the mammalian cell genome;
(b) a second DNA fragment which comprises at least one exon but not all of the exons of a gene encoding a first selectable marker protein; and
(c) a third DNA fragment which comprises a region encoding a second selectable marker protein that is different from the first selectable protein and provides for selection of a mammalian cell which has said marker plasmid integrated into its genome;
(ii) selecting a cell which contains the marker plasmid integrated in its genome by screening in vitro for expression of a selectable marker protein encoded by said third DNA fragment;
(iii) transfecting or transforming said selected cell with a target plasmid which comprises at least one DNA to be inserted into the genome of said cell, and further comprises the following sequences;
(a) a fourth DNA fragment which comprises a region that is identical or is sufficiently homologous to the unique site for homologous recombination in the marker plasmid such that this region can recombine with said marker plasmid DNA via homologous recombination; and
(b) a fifth DNA fragment which comprises the remaining exon or exons of the gene encoding a first selectable marker protein that are not present in the marker plasmid;
wherein an active first selectable marker protein is only produced if the at least one exon of a gene encoding a first selectable marker protein contained in the marker plasmid is expressed in association with the remaining exon or exons of the gene encoding a first selectable marker protein contained in the target plasmid; and
(iv) selecting cells which contain the target plasmid integrated at the unique site for homologous recombination by screening in vitro for the expression of the first selectable marker protein.
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Abstract
A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.
30 Citations
41 Claims
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1. A method for inserting a desired DNA at a target site in the genome of a mammalian cell which comprises the following steps:
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(i) transfecting or transforming a mammalian cell in vitro with a marker plasmid comprising the following sequences; (a) a first DNA fragment which comprises a region that is heterologous to the mammalian cell genome and provides a unique site for homologous recombination when it is integrated in the mammalian cell genome; (b) a second DNA fragment which comprises at least one exon but not all of the exons of a gene encoding a first selectable marker protein; and (c) a third DNA fragment which comprises a region encoding a second selectable marker protein that is different from the first selectable protein and provides for selection of a mammalian cell which has said marker plasmid integrated into its genome; (ii) selecting a cell which contains the marker plasmid integrated in its genome by screening in vitro for expression of a selectable marker protein encoded by said third DNA fragment; (iii) transfecting or transforming said selected cell with a target plasmid which comprises at least one DNA to be inserted into the genome of said cell, and further comprises the following sequences; (a) a fourth DNA fragment which comprises a region that is identical or is sufficiently homologous to the unique site for homologous recombination in the marker plasmid such that this region can recombine with said marker plasmid DNA via homologous recombination; and (b) a fifth DNA fragment which comprises the remaining exon or exons of the gene encoding a first selectable marker protein that are not present in the marker plasmid; wherein an active first selectable marker protein is only produced if the at least one exon of a gene encoding a first selectable marker protein contained in the marker plasmid is expressed in association with the remaining exon or exons of the gene encoding a first selectable marker protein contained in the target plasmid; and (iv) selecting cells which contain the target plasmid integrated at the unique site for homologous recombination by screening in vitro for the expression of the first selectable marker protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A vector system for inserting a desired DNA at a target site in the genome of a mammalian cell in vitro which comprises at least the following:
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(i) a marker plasmid comprising the following sequences; (a) a first DNA fragment which comprises a region of DNA that is heterologous to the mammalian cell genome and provides a unique site for homologous recombination when it is integrated in the mammalian cell genome; (b) a second DNA fragment which comprises at least one exon but not all of the exons of a gene encoding a first selectable marker protein; and (c) a third DNA fragment which comprises a region encoding a second selectable marker protein that is different from the first selectable protein and provides for selection of a mammalian cell in vitro which has said marker plasmid integrated into its genome; and (ii) a target plasmid which comprises at least one DNA to be inserted into the genome of said cell, and further comprises the following sequences; (a) a fourth DNA fragment which comprises a region that is identical or is sufficiently homologous to the unique site for homologous recombination in the marker plasmid such that this region of DNA can recombine with said marker plasmid DNA via homologous recombination; (b) a fifth DNA fragment which comprises the remaining exon or exons of the gene encoding a first selectable marker protein that are not present in the marker plasmid; wherein the at least one exon of a gene encoding a first selectable marker protein in the marker plasmid and the remaining exon or exons of the gene encoding a first selectable marker protein in the target plasmid together encode an active first selectable marker protein. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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Specification