Methods comprising apoptosis inhibitors for the generation of transgenic pigs

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Stem Cell Related Patent Number US5859309

Title:Vector for integration site independent gene expression in mammalian host cells
Inventors:Eccles, Sarah Jane; London, United Kingdom
Grosveld, Franklin Gerardus; London, United Kingdom
Summary:Described herein is a novel vector with an immunoglobulin dominant control region, and methods of its use in eliciting specific gene expression in mammalian host cells. The invention relates to an isolated DNA molecule comprised of a dominant control region that is functional in a mammalian cell and which permits immunoglobulin gene expression. Further disclosed are methods by which the molecule may be utilized as a vector 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, wherein the vector is described as comprising a promoter and the gene of interest and an immunoglobulin-dominant control region derived from the mouse lambda immunoglobulin gene locus which elicits host cell-type restricted, integration site-independent, copy number-dependent expression of the gene. Therapeutic and experimental applications are provided.
Abstract:A vector 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. The vector comprises a promoter and the gene and an immunoglobulin dominant control region derived from the mouse .lambda. immunoglobulin gene locus capable of eliciting host cell-type restricted, integration site independent, copy number dependent expression of the said gene. The DNaseI super hypersensitive site exemplified are i) about 2.35 kb upstream of the CAP site of the rearranged .lambda..sub.1 gene, ii) about 2.5 kb upstream of the genomic V.lambda..sub.2 segment or iii) about 30 kb downstream of the rearranged .lambda..sub.1 gene. Mammalian host cells transformed with the vector are disclosed as are transgenic mammals transformed with the vector and a method of producing a polypeptide comprising culturing a transformed mammalian cell. A method of gene therapy comprising the steps of i) removing stem cells from the body of a mammal, ii) optionally killing stem cells remaining in the body, iii) transforming the removed stem cells with the containing a gene deficient or absent in the body, and iv) replacing the transformed stem cells in the body is also disclosed. Also disclosed is functional mouse immunoglobulin .lambda..sub.1 enhancer consisting of a DNA sequence comprising all or a functional part of the DNA sequence between the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene and the SnaBI site 10 kb downstream of this Xho I site. The functional mouse immunoglobulin .lambda..sub.1 enhancer may comprise all or a functional part of i) the 1.3 kb first HindIII to HindII DNA fragment downstream of the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene, ii) the 3.3 kb HindII to HindII DNA fragment downstream of the EcoRI site 3.8 kb downstream of the Xho I site in the rearranged mouse .lambda..sub.1 gene and spanning the SnaBI site 10 kb downstream of this Xho I site.
US Patent Website:Click Here for Full Text of Patent
Title Number:US5859309
Application Number:US1995000481035
Date Filed:07/06/1995
Date Published:12/01/1999
Assignee:Medical Research Council, Ltd., London, United Kingdom


 
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