Method for concentrating cells that are genetically altered by nucleases
AssigneeToolgen Incorporated
InventorsJin Soo Kim, Seok Joong Kim, Yong Sub Kim
Life of this patent
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Abstract
The present invention relates to a reporter construct and method for identifying or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage; a host cell comprising the reporter construct; and a system for monitoring a nuclease activity. The reporter system of the present invention is simple and non-invasive, and allows for an efficient enrichment of the gene-modified cells. Therefore, the present invention will promote the application of a nuclease in the field of gene therapy and genetic engineering as well as basic research.
Claims17
A non-homologous end joining (NHEJ) reporter construct for identifying, selecting, or enriching cells that have been modified by NHEJ at a target sequence in the genome of the cells, the construct comprising in 5′ to 3′ order:
- a) a promoter,
- b) a first reporter gene operably linked to the promoter,
- c) the target sequence, wherein the target sequence is recognized by a nuclease specific for the target sequence, and
- d) a second reporter gene,
- wherein the second reporter gene is out of frame with the first reporter gene, such that in the absence of the nuclease only the first reporter gene is expressed and wherein in the presence of the nuclease, the target sequence is cleaved and NHEJ repair results in a frame shift mutation that renders the first reporter gene in frame with the second reporter gene such that both the first reporter gene and the second reporter gene are expressed from the promoter.
A non-homologous end joining (NHEJ) reporter construct for identifying, selecting or enriching cells that have been modified by NHEJ at a target sequence in a genome of the cells, the construct comprising in 5′ to 3′ order:
- a) a promoter,
- b) a first reporter gene operably linked to the promoter,
- c) the target sequence, wherein the target sequence is recognized by a nuclease specific for the target sequence,
- d) a second reporter gene, and
- e) a third reporter gene,
- wherein the second reporter gene is out of frame with the first reporter gene and the third reporter gene is out of frame with the first reporter gene, such that in the absence of the nuclease, only the first reporter gene is expressed and wherein in the presence of the nuclease, the target sequence is cleaved and NHEJ repair results in a frame shift mutation that renders the first reporter gene in frame with the second reporter gene and/or the third reporter gene such that the second reporter gene and/or the third reporter gene are expressed with the first reporter gene.
Dependencies are inferred from the claim text.