Construction of Spontaneous Tumorigenesis Mouse Model of Esophageal Cancer based on Trp53 Knockout and Ccnd1 Overexpression in Esophageal Epithelium

ZHANG Xiusen; ZHANG Xudong; JIN Xing; ZHANG Shilei; GUO Xiaoxi; CHEN Shiyu; YUAN Xiang; GAO Shegan

doi:10.15926/j.cnki.issn2096-7381.2025.04.001

Abstract：Objective To construct a mouse esophageal cancer model with Trp53 gene knockout and Ccnd1 overexpression using the Cre-loxP system and CRISPR/Cas9 technology.Methods We targeted the 2nd to 9th exons of the Trp53 gene in C57BL/6 mice,designed RNA target sites,and obtained sgRNA through in vitro transcription.The sgRNA was mixed with mRNA encoding Cas9 and microinjected into fertilized eggs to produce F0 generation mice,which were then bred to obtain Trp53flox/flox mice.Meanwhile,based on the Cre-loxP system,we designed a conditional overexpression mouse model of the Ccnd1 gene(Ccnd1R26-LSL-mcherry)at a safe locus in C57BL/6 mice.We mated Trp53fl/fl mice,Ccnd1R26-LSL-mcherry mice,and EDL2-Cre mice to produce offspring.We collected tail tissues from the mice,extracted DNA,and used PCR and agarose gel electrophoresis to genotype the offspring and verify the expression of the genes in the mice.We successfully obtained the target mice,namely esophageal epithelial Trp53 knockout and Ccnd1 overexpressing mice(EDL2-Cre;Trp53fl/fl;Ccnd1R26-LSL-mcherry mice,hereinafter referred to as CP mice).We extracted proteins from the esophageal tissues of CP mice and used Western blot to detect the expression levels of Trp53 and Ccnd1 proteins.We also recorded the body weight of CP mice and compared it with that of wild-type mice.Additionally,we collected mice at different growth stages and performed hematoxylin and eosin(HE)staining to observe the morphological structure of the esophageal tissues.Results Based on the DNA extracted from the tail of the mouse,the genotype of the mouse could be identified using PCR,and CP mice had been successfully bred.The mouse could grow normally and had no significant difference in weight compared to wild-type mice(P＞0.05).The mouse had good growth status and normal diet and drinking water.According to the statistical results of HE staining of esophageal slices after euthanasia of mice,the incidence of esophageal cancer could reach 28％at 12 months of age,which could be used for later research.Conclusion The successfully constructed CP mice exhibit esophageal epithelial-specific carcinogenesis during normal growth.This model provides an in vivo platform for exploring the pathophysiological mechanisms of esophageal cancer and the impact of the immune microenvironment on tumor development.