The Interactions Of Gene Silences Of STAT4, STAT6 And CBP In JAK-STATs Signal Pathway In Jurkat Cells

Objective: To transfect interference-plasmids to silence the genes of STAT4, STAT6 and CBP, and to observe the interactions between STAT4, STAT6 and CBP, the Jurkat celllines of overexpression of STAT4 and STAT6 are to be constructed.Methods: The plasmids pIRES2-EGFP, pIRES2-EGFP-STAT4 and pIRES2-EGFP-STAT6 which have Neo/Kan gene are transfected into Jurkat cells by DMRIE-C, and then use the screening-drug G418 to get celllines of overexpression of STAT4 and STAT6.Results:①The best ratioes of plasmids and DMRIE-C are changed when the cell density is different.②G418 500 mg/ml is the reasonable screening concentration when the planted density of Jurkat is 2.5×105/cm~2.③The GFP expressions are at their peaks at 3~4 day, 6~7 day and 9 day respectively when plasmids pIRES2-EGFP, pIRES2-EGFP-STAT4 and pIRES2-EGFP-STAT6 are transfected.④The transfection efficiencies of Group 2.5×105/cm~2 and Group 1.5×105/cm~2 are almost the same by iposome DMRIE-C.⑤After screened by G418, stable transfection cell-clones are got from Group 1.5×105/cm~2, while not from Group 2.5×105/cm~2.⑥After screened by G418, stable transfection cell-clones are got from the Jurkat cells transfected with plasmid pIRES2-EGFP-STAT6, not from the Jurkat cells transfected with plasmid pIRES2-EGFP-STAT4.Conclusion: Some features of expression of plasmids and experiences of stable transfection are got. What is more, cell-clones are got which are from Jurkat cells transfected with plasmid pIRES2-EGFP-STAT6, while no cell-clone is got when Jurkat cells are transfected with plasmid pIRES2-EGFP-STAT4.Part2 Optimization of Lipofectamine(?) LTX Transfecting Jurkat CellsObjective: It is to set up an optimized scheme to develop the transfection efficiency of Jurkat by Lipofectamine(?) LTX.Methods: The plasmids pIRES2-EGFP and pIRES2-EGFP-STAT6 which can express GFP and Lipofectamine(?) LTX were used to transfect Jurkat cells, investigating the influences of transient transfection from a series of factors, such as cells'state before transfection, ratio of liposome and DNA, cell density and different volumes of transfection media.Results:The growth rate of Jurkat is related to cell density, and renewing the culture media 1 day before transfection can ensure nice growth state of cells, while freshness of culture media has little or no effect on cells'growth state. The transfection efficiency is highest when the ratio of liposome and DNA is 2.75μL:500 ng. To improve the utilization of transfection reagent, it is important to increase density of Jurkat advisably. 48 h after transfection, the transfection efficiency of Group 300μL is as(1.4±0.2)times as Group 600μL, and the statistic difference is considered significient (P<0.05). 24 h after transfection, the transfection efficiency of different ratios of liposome and DNA is considered no significant statistic difference (P>0.05), while is significant 48 h after transfection (P<0.05), and transfection efficiency of the two plasmids is considered significient (P<0.05).Conclusion:The study provides some constructive suggestions on the optimized transfection of Jurkat by Lipofectamine(?) LTX, and explains some regular rules of expression of genes after transfection,supplying methods for transfecting suspension Cells. Object: To analyze the interactions between the transcription factor STAT4, STAT6 and CBP, gene silences are made use of to observe the corresponding effects.Methods: The plasmids pGenesil-3-shSTAT4, pGenesil-3-shSTAT6 or pGenesil-3-shCBP is transfected with the detecting plasmids pGL3-IL-4R and pCAT3-IFN-γinto Jurkat cells, and IL-12 and IL-4 are used to act the Jak-STATs pathway, then luciferase assay system and CAT ELISA are used to observe the activity of the target genes.Results: The gene silence of CBP can lead to the decline of the gene-promoter's activity of of IL4R and IFNG,and the results have statistical significance (P<0.05), while the results got from gene-silence of STAT4 and STAT4 have no statistical significance.Conclusion :①CBP acts as a crucial accessory molecule for activing the genes of IL4R and IFNG.②The expression of CBP in Jurkat cells is limited.③The construction of CBP in the cells of human and mice may be highly conservative.④The interaction between the STAT4 and STAT6 is unknown for the present.