Construction Of Gene Knockout Mice Models For Gastric Cancer Study

Objective Genetically engineered mice are very useful animal models in scientific research,especially in the field of cancer.IRX1 belongs to Iroquois homeobox gene family and inhibits gastric cancer cell growth,invasion and tumorigenesis.The DICER1gene encodes endonuclease III and plays a key role in micro RNA maturation.Micro RNAs are involved in a variety of biological processes,including the development of tumors. FBXW7 encodes ubiquitin ligase and plays a role in tumor inhibition by participating in ubiquitin proteinase degradation and DNA damage repair.In order to study the role of IRX1?DICER1 and FBXW7 in gastric cancer,the following study were carried out.(1)Construction of IRX1,DICER1 and FBXW7 knockout mouse models;(2)Spontaneous tumor observation and chemical carcinogenesis.Methods(1)We constructed Irx1 knockout mice using Irx1 chimeric mice and observe the homozygous knockout embryos development.(2)We constructed Dicer1 knockout mice conditionally in Villin positive expressed gastric progenitor cells using Lox P-Cre system.Dicer1^-/-,Dicer1^+/-and Dicer1^+/+mice were raised for 50 weeks,the body weight,survival time,spontaneous tumor formation rate and tumor type were detected.We used chemical carcinogen MNU to induce gastric cancer.Immunofluorescence was used to detect the expression of Villin and Cre.Immunohistochemistry was used to detect the expression of Dicer1.We detected the effect of Dicer1 deletion on micro RNA and m RNA expression profiles on gastric tissue of mice by high-throughput sequencing.(3)We constructed Fbxw7 knockout mice using Fbxw7 chimeric mice.Fbxw7^+/-and Fbxw7^+/+ mice were treated with MNU.The rate of gastric tumor and tumor type were detected.The level of proliferation and apoptosis were measured by Ki67 and Tunel assays.We detected the expression of Fbxw7 and c-Myc in the gastric cancer samples of mice and human by WB.The DNA damage repair response after MNU treatment was evaluated by immunofluorescence,WB and comet assay in two kinds of MEFs from Fbxw7^+/-and Fbxw7^+/+mice.Results(1)Genotype of Irx1 knockout mice was analyzed by PCR with two pairs of primers(Irx1,TDP).In Irx1^-/-mice,PCR product of Irx1 is 709bp,while the TDP exihibits no band.In Irx1^+/-mice,Irx1 is 709bp,TDP is 106bp.In Irx1^+/+mice,Irx1 exihibit no band,TDP is 106bp.(2)Genotype of Dicer1 knockout mice was analyzed by PCR with three pairs of primers(Dicer1,Cre,Vil-cre).In Dicer1^-/-mice,PCR product of Dicer1 is420bp,while the Cre/Vil-cre exihibits 233bp/1100bp.In Dicer1^+/-mice,Dicer1 is 420bp and 351bp,Cre/Vil-cre is 233bp/1100bp.In Dicer1^+/+mice,Dicer1 is 351bp,Cre/Vil-cre exihibits no band.After 50 weeks of observation,the mortality of Dicer1^-/-mice was significantly higher than that of Dicer1^+/-and Dicer1^+/+mice.The weight of Dicer1^-/-mice was lower than that of Dicer1^+/-and Dicer1^+/+mice.At 50 weeks of age,none of these three genotypes of mice were found spontaneous gastric cancer.Dicer1^-/-mice appeared cystic degeneration of renal tubules and goblet cells disappearance and adenoma in colon.Dicer1^-/-,Dicer1^+/-and Dicer1^+/+mice were treated with MNU and sacrificed at 35 weeks of age.We found the presence of co-localization of VILLIN and CRE on Dicer1^-/-mice gastric tumor,kidney and colon.We also found that Dicer1 expression was decreased in gastric tumor of Dicer1^-/-mice.RNAs of mouse gastric tumors were extracted for sequencing analysis of micro RNA and m RNA profils.Comparing the results of Dicer1^-/-and Dicer1^+/+mice in the control group,44 differentially expressed micro RNAs and 1020differentially expressed genes were found.(3)PCR was used to detect the genotype using DNA from the mouse tail of Fbxw7 knockout mice.PCR product of Fbxw7^+/+mice was359bp,and PCR products of Fbxw7^+/-mice were presented as 2 bands of 232bp and 359bp respectively.Fbxw7^+/-mice developed more gastric tumors than Fbxw7^+/+mice.The level of apoptosis in Fbxw7^+/-mice was significantly decreased.We found Fbxw7 expression was lower in human and mouse gastric cancer than the adjacent normal tissue,while c-Myc was the opposite status.After DNA damage caused by MNU,immunofluorescence of p H2AX showed delayed DNA repair in MEFs from Fbxw7^+/-mice than Fbxw7^+/+mice.The same tendency was also disclosed in Comet assay and Western Blot analysis.Conclusion Knockout mice are very useful in the study of the relationship between specific genes and gastric carcinogenesis.The homologous knockout of Irx1 gene causes embryo lethality.There is no spontaneous gastric carcinogenesis in conditionally Dicer1knockout mice in Villin positive expressed gastric progenitor cells.The incidence of gastric cancer induced by chemical carcinogen is higher in conditionally Dicer1 gene knockout than that of wild type mice.Fbxw7 heterozygous knockout mice disclosed a higher incidence of gastric cancer than wild type mice under the MNU exposure,which may be associated with Fbxw7 haploinsufficiency resulting in increased expression of downstream oncoprotein c-Myc and delayed DNA damage repair.