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US 6,586,250 B1Granted
Method of PCR-based gene targeting
AssigneeWashington University
InventorLarry E. Fields
Life of this patent
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Abstract
An improved method of gene targeting, referred to as PCR-based gene targeting is disclosed, which generates cell lines or mice in which at least one allele of a specific gene is disrupted by double homologous recombination of a PCR-derived targeting vector with chromosomal DNA. The method is especially applied to murine macrophage cytokine-inducible nitric oxide synthase (MøiNOS).
Claims4
A method of PCR-based gene targeting comprising:
- (a) obtaining a PCR-derived homologous genomic DNA fragment by applying long-range PCR to a murine genomic DNA template to generate a 7 kb genomic amplicon spanning exons 4 to 7 of the NOS2 gene locus consisting of a 1.1 kb short-arm spanning exons 6 to 7 and a 5.9 kb long-arm spanning exons 4 to 6 of said NOS2 gene locus by employing
- (i) a first oligonucleotide primer having a sequence specific for the 5′ end of the target region and a suitable restriction site added at its 5′ end and
- (ii) a second oligonucleotide primer having a sequence specific for the 3′ end of the target region and a suitable restriction site added at its 3′ end, whereby said primers bind to, respectively, the 5′ and 3′ ends of said target area,
- (b) producing a PCR-based targeting vector by
- (i) inserting the 1.7 kb pgk-neo positive selectable marker gene into said amplicon at a suitable restriction site within said target region and
- (ii) inserting the 2.6 kb pgk-neo negative selectable marker gene at the 5′ or 3′ end of said amplicon and then linearizing said targeting vector, and
- (c) thereafter transfecting said PCR-based targeting vector into ES cells whereby said targeting vector undergoes efficient homologous recombination into said gene locus.
4 more elements
A method of PCR-based gene targeting comprising:
- (a) obtaining a PCR-derived homologous genomic DNA fragment by applying long-range PCR to a murine genomic DNA template consisting of RAW 264.7 murine macrophage cell line to generate a large genomic amplicon spanning the exons of a gene locus of interest by employing
- (i) a first oligonucleotide primer having a sequence specific for the 5′ end of the target region and a suitable restriction site added at its 5′ end and
- (ii) a second oligonucleotide primer having a sequence specific for the 3′ end of the target region and a suitable restriction site added at its 3′ end, whereby said primers bind to, respectively, the 5′ and 3′ ends of said target area,
- (b) producing a aPCR-based targeting vector by
- (i) inserting the 1.7 kb pgk-neo positive selectable marker gene into said amplicon at a suitable restriction site within said target region and
- (ii) inserting the 2.6 kb pgk-tk negative selectable marker gene at the 5′ or 3′ end of said amplicon and then linearizing said targeting vector, and
- (c) thereafter transfecting said PCR-based targeting vector into ES cells whereby said targeting vector undergoes efficient homologous recombination into said gene locus.
4 more elements
A method of PCR-based gene targeting comprising:
- (a) obtaining a PCR-derived homologous genomic DNA fragment by applying long-range PCR to a murine genomic DNA template of RAW 264.7 murine macrophage cells to generate a large genomic amplicon spanning exons 4 to 7 of the NOS2 gene locus by employing
- (i) a first oligonucleotide primer having a sequence specific for the 5′ end of said amplicon and a ClaI restriction site added at its 5′ end and
- (ii) a second oligonucleotide primer having a sequence specific for the 3′ end of said amplicon and a NotI restriction site added at its 3′ end,
- whereby said primers bind to, respectively, the 5′ and 3′ ends of said amplicon,
- (b) producing a PCR-based targeting vector by
- (i) inserting the 1.7 kb pgk-neo positive selectable marker gene into said amplicon at a SacI restriction site at the 3′ end of exon 6 of said amplicon and
- (ii) inserting the 2.6 kg pgk-tk negative selectable marker gene at a ClaI restriction site at the 5′ end of exon 4 of said amplicon
- and then linearizing said targeting vector, and
- (c) thereafter transfecting said PCR-based targeting vector into ES cells whereby said targeting vector undergoes efficient homologous recombination into said NOS2 gene locus.
6 more elements
Dependencies are inferred from the claim text.