US 9,809,839 B2
Method for concentrating cells that are genetically altered by nucleases
Jin Soo Kim, Seoul (KR); Seok Joong Kim, Seoul (KR); and Yong Sub Kim, Busanjin-gu (KR)
Assigned to TOOLGEN INC., Seoul (KR)
Appl. No. 14/920
Filed by Jin Soo Kim, Seoul (KR); Seok Joong Kim, Seoul (KR); and Yong Sub Kim, Busanjin-gu (KR)
PCT Filed Feb. 22, 2012, PCT No. PCT/KR2012/001367
§ 371(c)(1), (2), (4) Date Oct. 22, 2013,
PCT Pub. No. WO2012/115454, PCT Pub. Date Aug. 30, 2012.
Claims priority of provisional application 61/445,346, filed on Feb. 22, 2011.
Claims priority of application No. 10-2011-0093704 (KR), filed on Sep. 16, 2011.
Prior Publication US 2014/0045176 A1, Feb. 13, 2014
Int. Cl. C12Q 1/68 (2006.01); G01N 33/50 (2006.01); C12N 15/64 (2006.01); C12Q 1/44 (2006.01); C12Q 1/02 (2006.01); C12Q 1/66 (2006.01); C12N 15/63 (2006.01)
CPC C12Q 1/44 (2013.01) [C12N 15/64 (2013.01); G01N 33/50 (2013.01)] 17 Claims
1. 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.