Construction,Breeding And Identification Of CPLA2α Gene Knockout Mice

Objective c PLA2α 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 PLA2αwas high expressed in breast and liver cancer tissues, c PLA2α played an important role in breast and liver cancer cell migration and invasion. We also found that c PLA2α was involved in liver cancer cell EMT. In order to systematically study the role of c PLA2α in carcinogenesis and development, we constructed c PLA2α gene knockout mice model via TALEN technology. Then we inbred and identificated the c PLA2α heterozygote mice to obtain nullizygous mice. Finally in vivo assays were conducted to confirm the role of c PLA2α in tumorigenesis and metastasis.Methods1. We analyzed c PLA2α 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 PLA2α nullizygous mice.5. The expression of c PLA2α 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 PLA2α in tumor growth and metastasis. DEN-induced liver cancer mouse model was constructed to study the role of c PLA2α in tumorigenesis.Results1. We selected the exon 3 of c PLA2α 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 PLA2α nullizygous mice were obtained after marking and identification.5. c PLA2α did not expressed in nullizygous mouse heart tissue, and c PLA2α 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 PLA2α 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 PLA2α knockout mice through TALEN technology,and obtained F1 heterozygous mice.2. c PLA2α nullizygous mice were obtained after breeding, which provide an important animal model for the research of c PLA2α 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 PLA2α 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 PLA2α.5. c PLA2α knockout could remarkably inhibit the tumor metastasis in vivo.DEN-induced liver cancer model was successfully constructed.