Construction,Breeding And Identification Of CPLA2? Gene Knockout Mice

Objective c PLA2? high-selectively hydrolyzes the membrane phospholipids, initiates AA and lysophospholipid cascade, which result in the release of a series of inflammatory cytokines, finally promotes inflammation. In our previous study, we found c PLA2?was high expressed in breast and liver cancer tissues, c PLA2? played an important role in breast and liver cancer cell migration and invasion. We also found that c PLA2? was involved in liver cancer cell EMT. In order to systematically study the role of c PLA2? in carcinogenesis and development, we constructed c PLA2? gene knockout mice model via TALEN technology. Then we inbred and identificated the c PLA2? heterozygote mice to obtain nullizygous mice. Finally in vivo assays were conducted to confirm the role of c PLA2? in tumorigenesis and metastasis.Methods1. We analyzed c PLA2? gene sequences, designed TALEN targeting vector, and assembled TALEN vectors through PCR and Golden Gate strategy.2. The m RNAs of TALEN vectors were injected into fertilized mouse eggs, positive F0 and F1 mice were obtained after surrogacy and hybridization with wild type mice.3. Large-scale breeding was carried out using F1 heterozygous mice and their offsprings.4. We extracted DNA from pup's toe, the genotype was identificated by PCR and sequencing. Finally,we got c PLA2? nullizygous mice.5. The expression of c PLA2? was tested in tissues of three kinds of mice through Western Blotting assay.6. After collecting the metabolites of three kinds of mice, UPLC/Q-TOF-MS and analysis of metabolomics data were conducted, looking for obvious differences among groups.7. Lewis lung carcinoma model was constructed to comfirm the effect of c PLA2? in tumor growth and metastasis. DEN-induced liver cancer mouse model was constructed to study the role of c PLA2? in tumorigenesis.Results1. We selected the exon 3 of c PLA2? gene as TALEN target and successfully constructed of TALEN-L and TALEN-R vectors.2. 3 positive F0 mice were obtained after microinjection, and 7 F1 heterozygous mice were got after hybridization of F0 and WT mice.3. During 8 months breeding, the F1 mice totally produced 226 offsprings by 34 times of parturition.4. 36 c PLA2? nullizygous mice were obtained after marking and identification.5. c PLA2? did not expressed in nullizygous mouse heart tissue, and c PLA2? level was lower in heterozygous mouse tissue compared to WT.6. Metabolomics results showed that sevarial metabolites such as AA, LPA, PG, LT and TX had obvious differences among three groups.7. In vivo assay demostrated that the c PLA2? knockout had no significant effect on tumor growth, whereas almost completely inhibited the metastasis to liver. Liver cancer were successfully induced by DEN in both WT and KO mice.Conclusions1. We successfully constructed c PLA2? knockout mice through TALEN technology,and obtained F1 heterozygous mice.2. c PLA2? nullizygous mice were obtained after breeding, which provide an important animal model for the research of c PLA2? function in cacinogenesis and development of cancer.3. The nullizygous mice were identificated and validated in gene level and protein leves respectively.4. The metabolomic analysis of c PLA2? knockout caused changes not only indirectly confirmed the success of knockout model, but also provided potential markers for the study of the physiological function of c PLA2?.5. c PLA2? knockout could remarkably inhibit the tumor metastasis in vivo.DEN-induced liver cancer model was successfully constructed.