Constructing And Exploring Eukaryotic Expression Of Murine Interferon α4b Reporter Gene

Interferon(IFN) is a kind of high biological activity glucoprotein produced by Interferon Producing Cells(IPCs) such as B cell, monocyte, Natural killer cell, dendritic cell and fibroblast et al., when they are stimulated by appropriate stimulators. Interferon can be divided into three types according to the difference of interferon signal transduction receptor compound and sequence homology. Among all the three types of interferon, typeâ… interferon is consisted of IFN-α, IFN-βand IFN-ω. And IFN-βand IFN-ωshare the same ligands , IFNAR1 and IFNAR2, with IFN-α. Sendai virus, New castle disease virus, poly I:C, dsRNA and CpG ODN et al. are most commonly used stimulators. Different types of stimulators activate interferon transcription factors which start the transcription of typeâ… IFN through two different signal transduction paths, which are TLR path and nucleic acid protein combining path. CpG ODN integrating with TLR9 activates interferon transcription factor through TLR path while supressive ODN restrains or decreases the production of IFN.The process of typeâ… IFN production ,which actually is a process of positive feedback, can be divided into two phases, the early phase and the later phase, acrroding to the output of IFN. The IPCs produce modest quantity of IFN-βand IFN-α4 in the early phase which will combine with IFNAR in the later phase. The combination of IFN-βand IFN-α4 with their receptor activates much IRF-7 (Interferon Regulation Factor-7) which will integrate with the promoter of IFN-βand IFN-αto start the transcription of IFN-βand all subtypes of IFN-αamong which the biggest output one is IFN-α4.Our lab designed a lot of CpG ODN and supressive ODN whose activities are different. We want to screen the ODN of higher activity than others. PBMC poliferation assay and VSV virus protection assay are used routinely for screening ODN so far. We would like to establish a experiment platform to screen ODN basing on the signal transduction route of IFN production. The activity of CpG ODN is parallelled with the output of IFN whereas the activity of supressive ODN is inversed with it. Therefore the output of IFN could mirror the activity of ODN.We constructed a recombinant eukaryotic expressing plasmid pcDNA3/ P_mIFN-α4b-GFP firstly. Second, we transfected the plasmid into murine interferon producing cell. At last, we screen for a stable cell line with the recombinant eukaryotic expressing plasmid pcDNA3/P_mIFN-α4b-GFP which is the experiment platform.This paper reported what we have done. We first designed the primers of coding genes of mIFN-α4b promoter and GFP using the plasmid pGL-3/PmIFN-α4 and pcDAN3-GFP as templates preserved in our lab which were proliferated the two gene segment of mIFN-α4b promoter and GFP. Second, we conducted PCR assay to proliferate the two genes using the primers we designed. Then the two coding genes were inserted into the eukaryotic expressing plasmid pcDNA3 by DNA recombining technology. At last, we identified the recombinant plasmid pcDNA3/P_mIFN-α4b-GFP and sequenced them. And found that the mIFN-α4b promoter coding gene and GFP coding gene were all correct and were inserted into the right place.So we constructed the recombinant eukaryotic expressing plasmid pcDNA3/ P_mIFN-α4b-GFP containing mIFN-α4b reportor gene. Then, we tried to establish an effective transient transfection platform through transfecting the control plasmid pcDAN3-GFP for plasmid pcDAN3-GFP got the CMV promoter which could start transcription without stimulation.At the very beginning, we prepared plasmid pcDAN3-GFP of high purity and concentration by TIANprep Mini kit which was then determined by the GIS gel analyzing system, and obtained that the concentration of plasmid pcDAN3-GFP was 533.085 ng/?L. Then we improved the tranfection effiency from the following three aspects to establish the transient transfection platform: target cell for transfection, transfection method and transfection system. We first chose Calcium Phosphate transfection method to transfect plasmid pcDAN3-GFP into NIH-3T3 cell in 96 well plate. We detected the number of cell expressing GFP protein by confocal microscopy and got that there was less than 10 % NIH-3T3 cell expressing GFP protein. So we switched to transfect NIH-3T3 cell by lipofectin transfection in 96 cell plate. After the cells were plated 24 h, we began transfection. We prepared transfection compound 50μL in the light of Instruction Manual of transfection reagent Lipofectamine TM 2000. The transfection compound was incubated for 30 min at room temperature, then dropped into the plate with cell washed by serum free culture for twice. Then put the plate into incubator (37°C, 5 % CO₂) . 5 h later, changed the 50μL transfection compound into 100μL 10% FBS culture and put the plate back into incubator (37°C, 5 % CO₂). Detected the number of cell expressing GFP protein after cultivated for 24 h. And attained that there was less than 10% NIH-3T3 cell expressing GFP protein. Considering the possible reason why the transfection efficiency was low was that we did not optimize the transfection system, we optimized the concentration of transfection compound. We plated NIH-3T3 cell in 96 well plate. Cultivated in the incubator for 24 h, then we began transfection. We prepared transfection compound 50μL in the light of Instruction Manual of transfection reagent Lipofectamine TM 2000 for Well A and 25μL for Well B. The transfection compound was incubated for 30 min at room temperature, then dropped into the plate with cell washed by serum free culture for twice. Added 50μL serum free culture to Well A while added 75μL. Then put the plate into incubator (37°C, 5 % CO₂) . 5 h later, changed the 100μL transfection compound into 100μL 10% FBS culture and put the plate back into incubator (37°C, 5 % CO₂). Detected the number of cell expressing GFP protein after cultivating the cell for 24 h. And found that there was less than 10 % NIH-3T3 cell expressing GFP protein. Thinking about the reason why the transfection efficiency was so low was that first, the transfection target cell NIH-3T3 cell was not in good order; second, it was not appropriate to conduct transfection in 96 well plate for the number of cell was too small. We transfected L929 cell in 6 well plate through lipofectin transfection and added G418 into the cell 24 h after transfection when the resistance gene (Neo) expressed to kill the cell without the plasmid pcDAN3-GFP. And we added G418 into L929 cells which were not transfected and got that the cells could be killed after the cell was cultivated with G418 for 24 h, when the concentration of G418 was 400μg/mL. We plated L929 cells into 6 well plate. After the cells were plated 24 h, we began transfection. We prepared transfection compound 500μL in the light of Instruction Manual of transfection reagent Lipofectamine TM 2000. The transfection compound was incubated for 30 min at room temperature, then dropped into the plate with cell washed by serum free culture for twice. Added 1.5 mL serum free culture into the well. Then put the plate into incubator (37°C, 5 % CO₂) . 5 h later, changed the 2 mL transfection compound into2 mL 10% FBS culture and put the plate back into incubator (37°C, 5 % CO₂). Added G418 to the cells with the concentration of 400μg/mL and put the plate back into the incubator. Detected the number of cell expressing GFP protein after cultivating the cell for 24 h. And found that there were more than 30 % NIH-3T3 cell expressing GFP protein.In conclusion, we constructed the recombinant plasmid pcDNA3/P_mIFN-α4b-GFP containing murine IFN-α4b reportor gene, which was the basement of the following experiment. When referred to the aspect of eukaryotic expressing of plasmid pcDNA3/P_mIFN-α4b-GFP, we proposed a method that adding G418 when the resistant gene expressed which could improve the transfection efficiency. At last, we established the transfection plateform which could be conducted as following. Transfect the positive control plasmid pcDAN3-GFP of plasmid pcDNA3/P_mIFN-α4b-GFP into L929 cells in 6 well plate through lipofectin transfection. Add G418 with a concentration of 400μg/mL after transfection for 24 h. Detect the numbers of cell expressing GFP protein by confocal microscopy after cultivating the cell with G418 for 24h.