Osteopontin Gene Silencing Mouse Model Made By Lentivirus-mediated RNA Interference Technology

Part one Construction and identification of lentivirus-based vectors of short hairpin RNA targeting osteopontin[Abstract] Objective To construct a short hairpin RNA(shRNA) sequence targeting osteopontin (OPN) gene, and synthesize pSicoR-GFP-shRNA lentivirus recombinant, then screen the most effective shRNA sequences silencing osteopontin gene expressio.Method We designed shRNA-A targeting OPN according to the sequence of osteopontin form GeneBank, and shRNA-NC targeting none of the genes of C57mouse, also shRNA-B referring to the relevant literature. The above shRNA sequences were inserted into linear pSicoR-GFP lentivirus vector respectively; and then they were transformed into E. Coli JM109competent cells. The colonies were examed by PCR and the positive colonies were sequenced. After identification of the sequences, the right one, coupled with plasmid pCMV-VSV-G and pCMV-dR8.91, were transfected to293FT cells for packaging of lentivirus. Finally the virus was successfully packaged with a titer controlled above1x109TU/ml. Mouse hepatocytes were infected by this lentivirus for screening of the most effective shRNA sequences. Results As is shown by qRT-PCR and Western-Blot results, OPN-shRNA recombinant lentivirus can effectively inhibit mouse hepatocyte OPN gene expressio. And the most effective one was pSicoR-GFP-OPN-shRNA-A, it is efficiency was up to80%-90%. Conclusion OPN shRNA recombinant lentivirus was successfully constructed, and it can significantly inhibit the expression level of OPN. This laid the foundation for further in vivo study.Part two Establishment of the C57mouse liver osteopontin protein gene silencing model[Abstract] Objective To establish osteopontin (OPN) gene silencing model by injecting the packaged lentivirus into C57mouse through tail vein. Methods30C57mice were randomly and meanly divided into three groups:Blank control group (BC): injected with sterilized PBS200ul via the tail vein; negative control group (NC): injected with200ul lentivirus pSicoR-GFP-shRNA-EC via the tail vein; experimental group (EG):injected with200ul lentivirus pSicoR the GFP-shRNA-A via the tail vein. At48hours,72hours,96hours,7days, and14days after injection, two mice of each group were sacrificed for the collection of liver. One parts were embeded with OCT immediately, and underwent frozen slice for the detection of liver lentiviral transfection efficiency, The remaining specimens were tested with qRT-PCR and Western-Blot for the detection of OPN gene silencing efficacy. Results Compared with blank control group, and negative control group, the levels of osteopontin mRNA and protein of experimental group were significantly decreased to70%-80%; and there was no diffence between blank control group and the negative control group. Conclusion Using the technology of RNA interference, with the help of lentivirus vector, the liver OPN gene silencing mouse model was successfully established.Part three Detection of the changes of liver cholesterol metabolism--related genes of OPN gene silencing C57mouse[Abstract] Objective To study the expression of cholesterol metabolism and transport related genes in C57mouse liver after the silencing of osteopontin (OPN), and provide technical support for further study of relation between OPN and hepatic cholesterol metabolism related genes in cholesterol gallstone disease. Methods RT-PCR and Western-Blot were employed to measure the expression of the mRNA and protein of liver cholesterol metabolism and transport related genes of HMG-CoA ruductase, CYP-7α1and ABCG5/8. Results As is shown by RT-PCR, the expression of HMG-CoA ruductase, CYP-7α1,ABCG5/8did not changes significantly, after the silencing of OPN gene in mouse liver, and this effect was confirmed by Western-Blot. Conclusion Through this experiment, we mastered the technic of measureing the mRNA and protein levels of HMG-CoA reductase, CYP-7α1, ABCG5/8by RT-PCR and Western-Blot. After the silencing of OPN gene in C57mouse liver, there were no obvious changes of HMG-CoA ruductase, CYP-7α1and ABCG5/8genes. Through further literature review, we conjecture that the reason of this phenomenon may be that:1The playing of OPN’s role is on the condition of the highly expression of OPN under the metabolism disorders of the body caused by other Pathogenic factors;2The exertion of regulating role on other releated gene of OPN may be required a longer time (probably2months)Summary, during this study, using lentivirus-mediated RNA interference technology, we successfully established low liver osteopontin expression C57mouse modle. This provides a powerful research tool for further study of the regulatory role of OPN in cholesterol metabolism during cholesterol gallstone formation.The study of the third part, not only laid the methodological foundation for follow-up experiments, but also prompt us that:for further research, gallstone stone model should be built by fed OPN low expression C57mouse with high-fat, high cholesterol lithogenic diet.