Method for establishing colorectal cancer p73 reporter gene cell line

• Main Authors: YU HONGBING, LIU XIN, HAN CUIFANG
• Format: Patent
• Language: Chinese; English
• Published: 21.05.2019
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

The invention discloses a method for establishing a colorectal cancer p73 reporter gene cell line. The method specifically includes the steps of designing the specificity sgRNA sequence of a p73 genetic locus, cloning a plasmid PX459, integrating the homologous recombination sequence of a p73 gene and a green fluorescent protein DNA segment (EGFP), converting the plasmid and the integrated segmentinto a colorectal cancer cell line HCT116 through electric shock, screening EGFP expression cells from single cells through a flow cytometer, amplifying a monoclonal cell line, verifying the positivep73 reporter gene cell line through PCR authentication and western blotting from the screened EGFP expression cell line. Through the common expression of the colorectal cancer cell line p73 gene andEGFP, the EGFP expression level of the cell line is the same as that of the p73 gene, and therefore the expression level of the p73 gene can be accurately judged by detecting the EGFP expression levelvariation. The cell line es