US5635355A
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Methods of obtaining a DNA sequence for integration site independent gene expression
Assignee: Medical Research Council
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Filing: 1995-06-07
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Granted: 1997-06-03
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Expires: 2015-06-07
Abstract
Abstract Vectors for the integration of a gene into the genetic material of a mammalian host cell such that the gene may be expressed by the host cell comprise a promoter and the gene and include a dominant activator sequence capable of eliciting host cell-type restricted, integration site independent, copy number dependent expression of the gene.
Inventors
- 👤 Franklin Grosveld
- 👤 Dimitris Kioussis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 03:42
CPC Classifications
A; A01; A01K; A01K67/00; A01K67/027; A01K67/0275; C; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/70503; C07K14/70507; C07K14/795; C07K14/805; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/87; C12N15/90; A01K2217/00; A01K2217/05; A61; A61K; A61K48/00
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
159 words
A method of obtaining a DNA fragment comprising a dominant activator sequence, comprising 1) providing a candidate DNA fragment comprising a DNase I hypersensitive site from a genetic locus containing a structural gene that is expressed in a manner that is specific for a particular mammalian cell type; 2) ligating the fragment to an expressible gene to form a construct; 3) introducing the construct into the genome of a host cell of said particular mammalian cell type; and 4) determining if expression of said gene in said particular mammalian cell type is (a) dependent on the number of copies of said gene that are integrated into said genome, in that said expression increases as said number of copies of said gene increases; and (b) independent of the integration site of said construct in said genome, wherein such copy number-dependent and integration site-independent gene expression is indicative of the presence of a dominant activator sequence in said candidate DNA fragment.
Claim 2
Independent Claim
159 words
A method of obtaining a DNA fragment comprising a dominant activator sequence, comprising 1) providing a candidate DNA fragment comprising a DNase I hypersensitive site from a genetic locus containing a structural gene that is expressed in a manner that is specific for a particular mammalian cell type; 2) ligating the fragment to an expressible gene to form a construct; 3) introducing the construct into the genome of a host cell of said particular mammalian cell type; and 4) determining if expression of said gene in said particular mammalian cell type is (a) dependent on the number of copies of said gene that are integrated into said genome, in that said expression increases as said number of copies of said gene increases; and (b) independent of the integration site of said construct in said genome, wherein such copy number-dependent and integration site-independent gene expression is indicative of the presence of a dominant activator sequence in said candidate DNA fragment.
Claim 3
Dependent Claim
23 words
The method of claim 1, further comprising the step of determining whether said DNA fragment of step contains a DNase I hypersensitive site.
Claim 4
Dependent Claim
24 words
The method of claim 2 wherein said determining step comprises performing DNase I digestion on said DNA fragment and detecting DNA after said digestion.
Claim 5
Dependent Claim
24 words
The method of claim 3 wherein said detecting comprises performing a Southern blot utilizing a DNA probe that is hybridizable with said DNA fragment.
Claim 6
Dependent Claim
25 words
The method of claim 1 wherein said introducing step comprises introducing said construct into a fertilized egg of an mammal to produce a transgenic mammal.
Claim 7
Dependent Claim
43 words
The method of claim 5 wherein said determining step comprises comparing expression of said transgene in said particular mammalian cell type of two transgenic mammals, wherein said two transgenic mammals differ in the number of copies of said transgene contained in their genomes.
Claim 8
Dependent Claim
29 words
The method of claim 6 wherein said determining step comprises comparing levels of RNA complementary to said transgene in said particular mammalian cell type of said two transgenic mammals.
References Cited (Backward Citations)
No citation records indexed.
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