Method for obtaining an enriched population of siRNA-expressing cells
AssigneeUniversity of Utah Research Foundation
InventorsVicente Planelles, Erik Zimmerman, Jason DeHart
Life of this patent
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Abstract
Problems with transience of siRNA-mediated knock-down and transfection efficiency have limited the scope of RNAi-based experiments. The invention provides a tool for employing RNAi more efficiently and effectively by integrating RNAi expression with methods of cell enrichment.
Claims25
A method of obtaining cells capable of expressing inhibitory RNA, the method comprising:
- introducing at least one nucleic acid into a cell, wherein the at least one nucleic acid is able to express an RNAi molecule and a separation marker;
- expressing a separation marker;
- sorting the cell based on expression of the separation marker;
- and
- expressing the RNAi molecule in an amount sufficient to inhibit expression of the target gene.
A method of enriching a population of mammalian cells having an inhibitory RNA sequence, the method comprising:
- providing mammalian cells containing a target gene, wherein target cells are susceptible to RNA interference, and the target gene is expressed in the target cells;
- introducing in the mammalian cells an RNA molecule, wherein the RNA molecule may produce RNA interference and contains a double-stranded area with a first region having a sequence which corresponds to a nucleotide sequence of the target gene and a second region having a sequence which is complementary to the first region, wherein the first and the second regions hybridize to each other to form a double-stranded RNA molecule;
- introducing in the mammalian cells a separation marker;
- expressing the separation marker in the mammalian cells, wherein expression of the separation marker is indicative of the presence of the double-stranded RNA molecule;
- and
- sorting the mammalian cells expressing the separation marker, thereby enriching for the mammalian cells having the double-stranded RNA sequence.
A recombinant nucleic acid sequence comprising at least one nucleic acid sequence, wherein the at least one nucleic acid sequence is capable of expressing a separation marker and the at least one nucleic acid sequence comprises a promoter operably linked to a sequence capable of producing an inhibitor RNA, wherein the inhibitor RNA comprises a sequence complementary to a target gene.
A recombinant nucleic acid sequence comprising:
- at least one nucleic acid sequence having means for producing a separation marker and means for producing a ribonucleic acid sequence which is able to inhibit translation of a target gene.
Dependencies are inferred from the claim text.