MEANS AND METHODS FOR shRNA MEDIATED CONDITIONAL KNOCKDOWN OF GENES
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United States of America Patent
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Issued Date -
Nov 25, 2010
app pub date -
Sep 17, 2008
filing date -
Sep 17, 2007
priority date (Note) -
Abandoned
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Abstract
The present invention relates to a combination of DNA segments comprising: (a) a first segment comprising in 5′ to 3′ or 3′ to 5′ order: (aa) a promoter; (ab) a first DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a first type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; (b) a second segment comprising in 5′ to 3′ or 3′ to 5′ order: (ba) a promoter; (bb) a second DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a second type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; wherein (i) said first type of recombinase recognition sequences are recognized and recombined by a recombinase but not recombined with said second type of recombinase recognition sequences; (ii) said second type of recombinase recognition sequences are recognized and recombined by the recombinase of (i) but not recombined with said first type of recombinase recognition sequences; and (iii) said DNA sequence of (ab) and (bb) is expressed under the control of said promoters of (aa) and (ba) upon removal of said transcriptional stop elements of (ab) and (bb) by the activity of a recombinase, resulting in transcription of said shRNA molecule in a cell. Further, the invention relates to a genetically engineered non-human animal and a method to produce said transgenic non-human animal. Also, the invention relates to a cell genetically engineered with the DNA molecule of the invention and a method of simultaneously knocking down two genes in a cell. Furthermore, envisaged is a method of identifying a combination of two target genes as a potential drug target and the use of the DNA molecule of the invention for the preparation of a composition for gene therapy.
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