Methods comprising apoptosis inhibitors for the generation of transgenic pigs

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Stem Cell Related Patent Number US5869248

Title:Targeted cleavage of RNA using ribonuclease P targeting and cleavage sequences
Inventors:Yuan, Yan; New Haven, CT, USA
Summary:This invention introduces a targeted ribonuclease P cleavage of RNA using an isolated oligonucleotide molecule comprising an external guide sequence. Central to the invention is the discovery that any RNA can be targeted for cleavage by RNase P from prokaryotic or eukaryotic cells using a suitably designed oligonucleotide to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNase P in vitro. Further disclosed are methods by which the EGS hydrogen bonds to the targeted RNA to form a partial tRNA-like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. Methods of modification of the EGS are provided, as are methods of combining it with an RNase P catalytic RNA sequence to form a single oligonucleotide molecule. Techniques are provided by which a suitable EGS or RIGS in vivo may be used to make a selected RNA a target for cleavage by a host cell RNase P or introduced RIGS, thus preventing expression of the function of the target RNA. Therapeutic applications are included in the treatment of cancer and bacterial infections, and in the prevention of the expression of a multitude of disease-causing genes in vivo.
Abstract:It has been discovered that any RNA can be targeted for cleavage by RNase P from prokaryotic or eukaryotic cells using a suitably designed oligonucleotide (external guide sequence, or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNase P in vitro. The EGS hydrogen bonds to the targeted RNA to form a partial tRNA like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. An EGS can be modified both by changes in sequence and by chemical modifications to the nucleotides. The EGS can be a separate molecule or can be combined with an RNase P catalytic RNA sequence to form a single oligonucleotide molecule (RNase P internal guide sequence or RIGS). Methods are also disclosed to randomly select and to express a suitable EGS or RIGS in vivo to make a selected RNA a target for cleavage by a host cell RNase P or introduced RIGS, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease- or disorder-causing genes in vivo.
US Patent Website:Click Here for Full Text of Patent
Title Number:US5869248
Application Number:US1996000702652
Date Filed:06/11/1996
Date Published:09/02/1999
Assignee:Yale University, New Haven, CT, USA


 
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