Process of knocking out Wnt3a gene and verification method thereof

Abstract

The invention discloses a process of knocking out Wnt3a gene and a verification method thereof. The knockout and verification of Wnt3a gene are finished through the following steps: establishment of a Cas9 lentiviral vector for Wnt3a gene, culture and passage of HepG2 cell, lentivirus infection and screening of target cell, verification of gene knockout efficiency through a mispairing enzyme method, cell protein analysis and cell proliferation detection by a CCK-8 method. The invention has the following advantages: the Wnt3a gene is knocked out by establishing a Cas9 double-vector lentivirus system for the first time; Crispr/Cas9 is a technology for accurately editing specific site of the genome of any species, and the cell-level single gene or multiple genes can be knocked out by the technology; compared with other gene editing technologies, the method has the advantages that the targeting accuracy is higher; only if the RNA target sequence is completely matched with the genome sequence, can the Cas9 cut the DNA and realize simultaneous knockout of multiple sites of the target gene; and moreover, the experimental period of vector establishment is short, the time and the cost are remarkably saved, and species limit is avoided.

Description

technical field [0001] The invention belongs to the medical field, and in particular relates to a process of knocking out the Wnt3a gene and a verification method thereof. Background technique [0002] Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors and ranks the third leading cause of cancer-related death. The occurrence and development of HCC is a complex process of multi-cause, multi-gene, multi-step and multi-factor coordination, involving multi-signal pathways and gene regulation such as activation of oncogenes, inactivation of tumor suppressor genes, and reactivation of some oncogenes during the embryonic period. Hepatocellular carcinoma is mainly treated with surgery, supplemented by chemotherapy and local treatment. Resection and liver transplantation are options for early liver cancer, but most patients have already lost the opportunity for surgical treatment when they see a doctor. Therefore, early diagnosis and...

AI Technical Summary

Problems solved by technology

Resection and liver transplantation are options for early liver cancer, but most patients have already lost the opportunity for surgical treatment when they see a doctor

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