Sirna expression system
AssigneeUniversita' Degli Studi Di Roma “La Sapienza”
InventorsIrene Bozzoni, Michela Denti
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Abstract
The authors describe an expression system for the correct, stable and effective expression in mammalian cells of a siRNA comprising: a) a polymerase H RNA-dependent promoter derived from the U1 RNA gene; downstream from the sequence b) suitable restriction sites for the cloning of the sequence transcribing the pre-siRNA; downstream from these sites c) sequences derived from the sequences at 3′ of the gene for U1 snRNA that are necessary and sufficient for the correct formation of 3′ of the presiRNA.
Claims8
A recombinant vector for the correct, stable and effective expression in mammalian cells of a siRNA or a miRNA, comprising from 5′ to 3′:
- a) an RNA polymerse II dependent promoter sequence derived from the U1 snRNA gene;
- b) suitable restriction sites for cloning the sequence that transcribes a presiRNA or a pre-miRNA;
- c) a sequence transcribing the pre-siRNA comprising:
- in position +1 an A or a G residue;
- a sequence from 21 to 23 nucleotides corresponding to a sense region of the mRNA transcribed by the gene to be silenced, that constitutes the first segment of the stem of the pre-siRNA;
- a sequence selected form a pre-miRNA sequence that constitutes the loop region of the pre-siRNA;
- a sequence from 21 to 23 nucleotides corresponding to the antisense region of the mRNA transcribed by the gene to be silenced that constitutes the second segment of the stem of the pre-siRNA;
- two final residues UU protruding in such a way that the following structure is obtained:
- or alternatively a sequence transcribing the pre-miRNA;
- d) termination sequences derived from the sequence at 3′ of the gene for U1 snRNA which are necessary and sufficient for the correct formation of the 3′ of the pre-siRNA or of the pre-miRNA.
Use the vectors according to any of claims from 1 to 6 for therapeutical applications.
Dependencies are inferred from the claim text.