Construction method of monoclonal cell line with specific gene knockout of porcine small intestine cells

• Main Authors: WANG YAO, ZHANG KUNPENG, MA SHENGMING, LIAN KAIQI, ZHANG WENHUI, WENG SHAOTING, WU YINRONG, LUO ZHONGYI
• Format: Patent
• Language: Chinese; English
• Published: 14.05.2024
• 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; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

The invention belongs to the technical field of gene engineering, and discloses a construction method and application of a monoclonal cell line with a specific gene knocked out of porcine small intestine cells, firstly, a gene editing vector is constructed, and gene editing can be carried out aiming at specific sites of genes; the rAAV-SaCas9-eGFP-sgRNA virus with strong small intestine tropism is packaged, so that the gene editing efficiency of cells is improved; and screening to obtain the specific gene knockout cell model of the porcine small intestine cells through rapid identification of eGFP fluorescence by flow cytometry. And a single cell strain is obtained by a cloning method, so that the monoclonal cell line with the specific gene knocked out of the porcine small intestine cells is obtained. According to the rAAV-SaCas9-eGFP-sgRNA virus disclosed by the invention, porcine small intestine cells are infected, and a method for rapidly establishing a monoclonal cell line with a specific gene knocked out