171 results about "Stable cell line" patented technology

The invention relates to an endogenous protein marking method applied to genome-wide binding spectrum analysis. The method comprises the steps of designing sgRNA for upstream and downstream 50bp regions of an encoding termination site of an NCOA1 gene; verifying cutting efficiency of sgRNA mediated Cas9 by an experiment; building an anti-estrogen therapy tolerant MCF-7 breast cancer cell model; by utilizing a CRISPR/Cas9 homologous recombination technology, selecting and using effective sgRNA and DNA donors, and introducing a Flag tag in the 3' end of the ER alpha co-transcription factor NCOA1 gene; and establishing a stable cell line with the Flag tag. Compared with the prior art, the method has the advantages that gene editing is performed by utilizing the CRISPR/Cas9 technology, so that the tag insertion efficiency of the gene editing is greatly improved; more importantly, chromatin structure parameters are introduced for designing sgRNA, so that effective cutting of Cas9 at a target site of a genome is ensured; and positive clone screening is performed by adopting DNA sequencing and RT-PCR sequencing methods, so that the positive clone identification step is simplified and the manpower and material resources for screening are reduced.