Rat Protein Arginine Methyltransferase Enzyme A Short Hairpin Rna Plasmid, Identification And In Vitro Rna Interference

PartⅠConstruction and identification of plasmid pGenesil-1-shRNA expressing the rat protein arginine methyltransferase 1Objective: To construct the expression plasmids of short hairpin RNA (shRNA) targeted to the rat protein arginine methyltransferases 1(PRMT1) gene for further investigating the relationship between the expression of PRMT1 gene and the function of vascular endothelium.Methods: The sequence of rat PRMT1 mRNA was found in the GenBank and copied to the design software at net. Two target sequences were selected. Single strands of encoding shRNA were designed , synthesized and conjuncted by annealing, after which the annealed duplexes were conjugated with plasmid vectors pGenesil-1 that were on linearization through digested with BamHⅠand HindⅢenzyme , generating the constructed pGenesil-1-PRMT1-shRNA. Recombinant plasmids were filted by cultured at LB plate containing kanamycin resistance. After these plasmids were extracted, they were identified with the digestion of Sal I enzyme determined sequence , and the inserted sequences were carried on DNA sequencing.Results: The constructs were all verified to fulfil design requirements through digested with Sal I enzyme. The inserted sequences were verified by DNA sequencing. The recombined shRNA plasmids was certified to be in the right rank.Conclusion: This experiment laid the foundation for further research on RNA interfere of PRMT1 gene in vitro.PartⅡOptimization of transfection conditions for transfecting pPRMT1–shRNA to rat arterial endothelial cellsObjective: To optimize the transfection parameters for transfecting the expression pPRMT1 -shRNA to rat arterial endothelial cells in primary culture mediated by polyethyleneimine jetPEITM-RGD.Methods: The primary culture of rat arterial endothelial cells was performed using the method of attachment block. The cells were identified with immunohistochemical SP method. Cells of the 3rd to 4th passages were used in the study. The complex was transfected at the different proportion of jetPEITM-RGD and pPRMT1–shRNA.24h after tranfection, the cells transfected of each ratio were counted under fluorescent microscope to determine the percentage. The cell viability was calculated using MTT assay at the same time.Results: The transfection efficiency was highest 53.54% when 3.0μg pPRMT1–shRNA was added into each well of 6 well cell culture plate and the N/P ratio equaled to 5. Conclusion: This experiment laid the foundation for performing efficiently cell transfection and further research on the effect of RNA interfere of PRMT1 gene on the plasma levels of homocysteine and asymmetric methylarginine and the function of vascular endothelium. PartⅢEffect of pPRMT1-shRNA on PRMT1 expression of rat arterial endothelial cells in vitroObjective: To investigate the effect of pPRMT1-shRNA on the expression of PRMT1 message RNA (mRNA) in rat arterial endothelial cells in vitro.Methods:Each plasmid of PRMT1-shRNA1, PRMT1-shRNA2 or HK was transfected into rat arterial endothelial cells. A blank control group was set up to be treated without any intervention at the same time, The cells of each group were collected separately12 and 24 hour after transfection. Total RNA was extracted separately from cells of each group with Trizol, and cDNA was constructed by reverse transcriptional PCR method. RT-PCR product were detected by 2% agarose gel electrophoresis. The Expression of PRMT1 Gene mRNA was analysed with gel analysis system.Result: The PRMT1 Gene mRNA level of pPRMT1-shRNA1 group and pPRMT1-shRNA2 group were significantly reduced than that of pHK group and of the blank control group at 12 and 24 hour after transfection(P<0.01); The suppression degree of PRMT1 Gene Expression of pPRMT1-shRNA1 group and pPRMT1-shRNA2 group at 12 hour was relatively significant than that at 24 hour after transfection(P<0.05); The suppression degree of PRMT1 Gene Expression of pPRMT1-shRNA1 group was more significant than that of pPRMT1-shRNA2 group(P<0.05). However, there was no significant difference in the PRMT1 Gene mRNA expression of pHK group and of the blank control group between 12 and 24 hour after transfection, and no significant difference between pHK group and the blank control group in the same time.Conclusion: pPRMT1-shRNA1 and pPRMT1-shRNA2 can inhibit efficiently and specifically PRMT1 Gene mRNA expression in vitro, which lays the foundation for further experiment in vivo.