Methods for producing microRNAs
The invention relates to recombinant vectors for inducible and/or tissue specific expression of double-stranded RNA molecules that interfere with the expression of a target gene. In certain embodiments, the invention relates to the use of Tet (tetracycline)-responsive RNA Polymerase II (Pol II) promoters (e.g., TetON or TetOFF) to direct inducible knockdown in certain cells of an integrated or an endogenous gene, such as p53. The invention also relates to a method for producing transgenic animals (e.g., mice) expressing inducible (such as tetracycline-regulated), reversible, and/or tissue-specific double-stranded RNA molecules that interfere with the expression of a target gene.
1. A non-human mammal comprising a population of cells, wherein each cell in said population harbors only a single, stably integrated copy of a nucleic acid construct for expression of a synthetic miR30-based shRNA, wherein said construct contains only a single tetracycline responsive RNA Polymerase II (Pol II) promoter operably linked to a sequence encoding only a single synthetic mir30-based shRNA directed against a target gene, wherein in said population of cells, induced expression of the synthetic miR30-based shRNA is sufficient to inhibit expression of the target gene by at least about 80%.
2. The non-human mammal of claim 1 , wherein the promoter comprises a tet-off promoter, such that the synthetic miR30-based shRNA is expressed in the absence of tetracycline or a tetracycline analog.
3. The non-human mammal of claim 1 , wherein the promoter comprises a tet-on promoter such that the synthetic miR30-based shRNA is expressed when the mammal is treated with tetracycline or a tetracycline analog.
4. The non-human mammal of claim 1 , 2 or 3 , wherein the population of cells further comprises an additional nucleic acid construct encoding tetracycline-controlled transactivator (tTA) or reverse tetracycline-controlled transactivator (rtTA).
5. The non-human mammal of claim 1 , 2 or 3 , wherein the population of cells further comprises an additional nucleic acid construct encoding Cre recombinase.
6. The non-human mammal of claim 1 , 2 or 3 , wherein the single tetracycline responsive RNA Polymerase II (Pol II) promoter is a LoxP-stop-LoxP system promoter.
7. The non-human mammal of claim 1 , 2 or 3 , which is a chimeric mammal, whose somatic or germ cells comprise said population of cells.