Sirna mediated gene silencing in transgenic animals
AssigneeArtemis Pharmaceuticals GmbH
InventorsJost Seibler, Frieder Schwenk, Ralf Kühn, Birgit Küter-Luks
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Abstract
The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.
Claims32
1-13. (canceled)
A method for constitutive and/or inducible gene knock down in a vertebrate, or in a tissue culture or cells of a cell culture derived from a vertebrate, which method comprises stably integrating an expression vector comprising a short hairpin RNA construct under control of a ubiquitous promoter into the genome of the vertebrate, of the tissue culture or of the cells of the cell culture.
The method of claim 1, wherein the expression vector is suitable for stabile integration into the genome of a vertebrate, or into the genome of the tissue culture or of cells of the cell culture.
The method of claim 1, wherein the expression vector contains homologous sequences suitable for integration at a defined genomic locus through homologous recombination in the genome of the vertebrate, in the genome of the tissue culture or in the genome of the cells of the cell culture.
The method of claim 3, wherein the cells of the cell culture are embryonic cells.
The method of claim 3 wherein the homolgous sequences are suitable for integration at a polymerase II dependent locus in the genome of the vertebrate, in the genome of the tissue culture or in the genome of the cells of the cell culture.
The method of claim 1, wherein the expression vector further contains functional sequences selected from the group consisting of splice acceptor sequences, polyadenylation sites and selectable marker sequences.
The method of claim 5, wherein the polymerase II dependent locus is selected from the group consisting of a Rosa26, collagen, RNA polymerase, actin and HPRT locus.
The method of claim 1, wherein the ubiquitous promoter is selected from the group of promoters consisting of polymerase I, II and III dependent promoters.
The method of claim 8 wherein the ubiquitous promoter is selected from the group consisting of a polymerase II or III dependent promoter.
The method of claim 8 wherein the ubiquitous promoter is selected from the group consisting of a CMV promoter, a CAGGS promoter, a snRNA promoter, a RNAse P RNA promoter, a tRNA promoter, a 7SL RNA promoter and a 5 S rRNA promoter.
The method of claim 10, wherein the S nRNA promoter is a U6 promoter.
The method of claim 10, wherein the RNAse P RNA promoter is a H1 promoter
The method of claim 1, wherein the ubiquitous promoter is a constitutive promoter.
The method of claim 1, wherein the ubiquitous promoter is an inducible promoter.
The method of claim 1 wherein said vertebrate is a non-human vertebrate.
The method of claim 1, wherein the expression vector is a Pol III dependent promoter driven shRNA construct suitable to be integrated into a ubiquitously active Pol II dependent locus;
The method of claim 1, wherein the expression vector is a Pol III dependent promoter driven shRNA construct suitable to be integrated into a ubiquitously active Pol II dependent locus.
The method of claim 1, wherein the expression vector is a Pol II dependent promoter driven shRNA construct suitable to be integrated into a ubiquitously active Pol II dependent locus.
The method of claim 1, wherein the shRNA comprises
- (I) at least one DNA segment A-B-C wherein
- A is a 15 to 35, preferably 19 to 29 bp DNA sequence with at least 95%, preferably 100% complementarily to the gene to be knocked down;
- B is a spacer DNA sequence having 5 to 9 bp forming the loop of the expressed RNA hair pin molecule, and
- C is a 15 to 35, preferably 19 to 29 bp DNA sequence with at least 85% complementarily to the sequence A, and
- (II) a stop and or polyadenylation.
The method of claim 1, wherein the expression vector is integrated at a polymerase dependent locus of the living organism, tissue culture or cell culture.
The method of claim 1, wherein the method for constitutive and/or inducible gene knock down in a vertebrate comprises integrating the expression vector into ES cells of the vertebrate.
A vertebrate, or tissue or cell culture derived from a vertebrate having stably integrated, preferably at a polymerase II dependent locus of the vertebrate, tissue culture or cells of the cell culture, an expression vector comprising a short hairpin RNA construct under control of a ubiquitous promoter.
An expression vector comprising a short hairpin RNA construct under control of a ubiquitous promoter.
Dependencies are inferred from the claim text.