Generation Of CRISPR/Cas9-Mediated ApoE And LDLR Double Gene Knockout Pigs As Atherosclerotic Models

Background: Atherosclerosis is mainly caused by the imbalance of blood lipids,especially in low density lipoprotein(LDL)metabolism.Apolipoprotein E(ApoE)and LDL receptor(LDLR)gene mutations play an important role in the development of atherosclerosis.Deficiency of ApoE can cause type ? hyperlipoproteinemia(HLP?).LDLR is situated on the surface of liver cells and related to cholesterol metabolism.Elimination of LDLR gene would lead to excessive accumulation of cholesterol in the blood circulation,and be associated with familial hypercholesterolemia(FH).Although some animals were widely treated as atherosclerotic models,such as rats,mice and hamsters,the anatomic and physiologic characteristics of those animal models are quite different from those of humans.Rats can not develop atherosclerosis spontaneously,and the plaque does not rupture after forming the plaque.At the same time,pigs are widely used in human disease research.Miniature pigs have high similarity with humans in terms of coronary anatomy,hemodynamics,and blood lipoprotein expression profiles.It can simulate the characteristics of human disease.Minipigs are ideal test animals for the construction of large animal models of atherosclerosis.Objective: The aim of this study is to generate LDLR/ApoE double gene knockout miniature pig by CRISPR/Cas9 technology,which might develop AS spontaneously without induction of high-fat diet,and maybe provide ideal materials for investigation of atherosclerotic pathogenesis.Methods: 1.According to NCBI,the LDLR and ApoE gene sequences of pigs were searched,and CRISPR/Cas9 targeting sites were designed at the corresponding sites.Build CRISPR/Cas9 targeting plasmids.2.The target plasmids were co-transfected with G418-resistant tdTomato plasmid into the primary fibroblasts of Bama pigs.After drug screening,monoclonal cells were picked and the cells' genetypes were determind by sequencing.3.The LDLR/ApoE double knockout cells were used as nuclear doners for porcine somatic cell nuclear transfer(SCNT),and the reconstructed procine embryos were transferred to the uterus of surrogate sows at the stage of blastocyst.4.The surrogate sows produced healthy clones of Bama pigs,fresh pig ear tissue was harvested,and genomes were extracted for genotyping.Blood lipids of neonatal LDLR/ApoE knockout pigs were detected by biochemical analyzer.The expression of LDLR and ApoE protein was detected by western-blot.Results: LDLR,ApoE CRISPR/Cas9 target plasmids were successfully constructed.After transfection of fibroblasts,a total of 2 female monoclonal LDLR/ApoE knockout cells were obtained,and 12 monoclonal cells were obtained from male cells.Porcine SCNT was successfully performed using identified cells as donors,and9 female and 33 male LDLR/ApoE double gene knockout pigs were produced.The results showed that the level of LDL-c and TC was significantly higher in LDLR/ApoE double gene knockout pigs than in wild type pigs(P < 0.05).Conclusion: In this study,LDLR /ApoE DKO atherosclerotic pigs were successfully obtained using CRISPR/Cas9 gene editing system combined with SCNT technology.Our results confirmed that the CRISPR/Cas9 gene editing system can efficiently modify the porcine genes,and LDLR /ApoE DKO pigs provide a good research vehicle for the further study of the mechanism of atherosclerosis.Background:Glioma,the most common primary central nervous system tumor,is one of the major intracranial tumors that endangers people's health.Currently,the treatment of gliomas is mainly surgical.However it is difficult to to achieve complete resection except the early and located in the appropriate part of tumors.Therefore,there is currently no safe and effective treatment for glioma.DHA is an important member of the omega-3 polyunsaturated fatty acid family and plays an important regulatory role in cell growth and maintenance.Studies have found that DHA can inhibit neuroblastoma cancer cells.This suggests that DHA may become a new drug for the treatment of neuroblastoma or other cancers.However,the molecular mechanism of DHA modulating gliomas is not clear.Prior to the advent of high-throughput sequencing technologies,the study of tumorigenesis was limited to one gene or several genes,and the development of tumors could not be systematically studied.In recent years,the development of RNA-sequencing(RNA-seq)technology has provided technical support for the overall development of tumors.Previous studies have suggested that cancer is a genetically-induced disease.However,recent studies have found that abnormalities in epigenetic levels of genes play an important role in the development of tumors.Long non-coding RNA(Lnc RNA)and micro RNA(mi RNA)are the two most studied RNAs with regulatory functions.In recent years,more and more studies have found that Lnc RNA is involved in the regulation of tumor cells.Co-expression network analysis revealed a significant correlation between changes in Lnc RNA expression levels and the expression of core cancer genes.This suggests that Lnc RNA can be used as a new target for the treatment of cancer.However,it is still unclear which genes DHA specifically regulates gliomas,and whether DHA regulates gliomas by affecting Lcn RNA expression.Therefore,this study will use transcriptome sequencing to screen differentially expressed genes and Lnc RNAs after treatment of glioma cells with DHA.Objective: To investigate the molecular mechanism of the regulation of proliferation,apoptosis,invasion and metastasis of glioma U251 cells by DHA.Methods: CCK-8 kit was used to detected the effect of DHA on proliferation of U251 cells.Transwell assay was used to detect the effect of DHA on the migration and invasion of U251 cells.Flow cytometry was used to detect the effect of DHA on the apoptosis of U251 cells.After U251 treatment with DHA,total cellular RNA was extracted for transcriptome sequencing.Then the sequencing results were correlated with bioinformatics analysis.Results: CCK-8 experiments showed that the inhibitory effect of DHA on U251 cell activity was dose-and time-dependent.DHA could significantly inhibit the migration and invasion of U251.Moreover,Flow cytometry results showed that DHA could significantly increase the apoptosis rate of U251.Transcriptome sequencing revealed significant changes in the expression levels of 293 genes.Classification of GO functions from biological processes,cell components,and molecular functions.The results showed that differentially expressed genes were mainly related to extracellular regions,cell adhesion,neurocognition,and receptor binding.KEGG analysis found that the most abundantly enriched pathways for these differentially expressed genes are metabolic,glioma-related pathways.q RT-PCR results showed that the differentially expressed genes were consistent with the sequencing results.In addition,DHA is also involved in the regulation of Lnc RNA expression in U251.A total of 75 lnc RNA expression levels were significantly changed.GO analysis showed that the target genes of differentially expressed Lnc RNAs are mainly related to intracellular components,metabolism,and molecular binding.KEGG analysis found that these target genes are mainly related to metabolism,apoptosis,tumorigenesis and other pathways.Conclusion: This study demonstrated that DHA has a significant inhibitory effect on the virulence of U251 cells.Through transcriptome sequencing,differentially expressed genes were screened after treatment with DHA.To provide a theoretical basis for further study of the mechanism and treatment of malignant glioma.