Gene loop induction expression regulating method
A technology of induced expression and looping, applied in biochemical equipment and methods, viruses/phages, nucleic acid vectors, etc., can solve the problems of long cycle, high price, and high cost of new drugs
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Embodiment 1
[0106] Embodiment 1, the construction of gene circuit induced expression regulation system (gene switch)
[0107] The gene loop induced expression regulation system and regulation method of the present invention, such as figure 1 shown. The construction and sources of the main plasmids are shown in Table 1. Specifically, the system includes: an initiation device (oleanolic acid (OA) or ursolic acid (UA)), a signal sensing device (GpBAR1), a signal processing device (TetR-hCREB1), a signal reporting effect device (P hCMVmin* -Reporter), close the device (DOX).
[0108] The gene loop induced expression regulation system (gene switch) of the present invention can realize effective implementation and regulation in vivo and in vitro, specifically as follows:
[0109] (1) In vitro experimental scheme, the specific steps are as follows:
[0110] The first step is to plant cells. Collect the collected cells at 3x10 per well 4 -5x10 4 Cells were planted in a 24-well plate, and 5...
Embodiment 2
[0127] Example 2, verifying that OA activates the GPBAR1 receptor, thereby activating the cAMP signaling pathway
[0128] The first step is to plant cells. The collected HEK-293T cells were collected at 5x10 per well 4 cells, seeded in a 24-well plate,
[0129] Add 500 μL / well of DMEM medium containing 10% FBS.
[0130] The second step is transfection. Transfection was carried out within 12 to 24 hours after the cells were planted (to ensure that the cells were adhered to the wall and in good growth condition), and the plasmid pXS25(P CMV -GpBAR1-pA), pXS13(P CMV -TetR-linker-hCREB1-pA), pMF111 (P TetO7 -SEAP-pA) in the optimal ratio (1:20:20) and PEI transfection reagent (mass ratio of plasmid to PEI 1:3) plus serum-free DMEM were mixed, the total volume was 50 μL / well, and stood at room temperature for 15 minutes Then evenly drop into the culture plate.
[0131] The third step is to add medicine. Change the medium 6 hours after transfection, add 500 μL / well DMEM medi...
Embodiment 3
[0133] Example 3, verifying the uniqueness of OA activating GPBAR1 to exercise the cAMP signaling pathway
[0134] The steps of this embodiment refer to the first and second steps in Example 2,
[0135] The third step is to add medicine. After 6 hours of transfection, the medium was changed, and 500 μL / well of DMEM medium containing 10% FBS was added. The medium was pre-mixed with a certain concentration of OA (20 μM) and different concentrations of PKA inhibitor H89
[0136] The fourth step is detection. After 48h, the reporter gene SEAP was detected. See the experimental results image 3 .
[0137] The experimental results and accompanying drawings of this example show that after OA activates GPBAR1, the cAMP signaling pathway is relatively uniquely activated, while other pathways are relatively insulated, indicating that the gene circuit designed in the present invention has high specificity.
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