@@ -102,10 +102,6 @@ Regulation System for Modulating Expression Levels</a>
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To obtain the results, we observed GFP fluorescence under 488nm excitation and 560nm emission using an inverted fluorescence microscope. We found that only the experimental group co-transfected with all three plasmids and irradiated with UV-B showed significant fluorescence, while all other groups showed no significant fluorescence. Some control groups, such as the group with the target gene plasmid + UVR8-VP64 plasmid and irradiated with UV-B, showed a small amount of fluorescence, which we consider to be gene expression leakage within an acceptable range. Images are as follows:
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In the previous pre-experiment, we verified RUP2 expression by Western blot and obtained the optimal concentration of 10 μg/ml for tetracycline induction by setting the concentration gradient. Plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag were simultaneously transfected into 293t cells. Transfection experiments were performed according to the following timeline, and the slides were observed under a confocal microscope after sealing.
<figcaptionstyle="width:70%">Figure 3 10x microscopic images of cells transfected with different plasmids with and without UV-B irradiation treatment. For cells transfected with each plasmid combination, three fields of view are selected for observation.
A. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP, pSNV2_VP64_NLS_coplat and pIRESN2_Gal4_UVR8at.
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@@ -113,6 +109,10 @@ Regulation System for Modulating Expression Levels</a>
C. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP, and pSNV2_VP64_NLS_coplat.
D. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP.
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In the previous pre-experiment, we verified RUP2 expression by Western blot and obtained the optimal concentration of 10 μg/ml for tetracycline induction by setting the concentration gradient. Plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag were simultaneously transfected into 293t cells. Transfection experiments were performed according to the following timeline, and the slides were observed under a confocal microscope after sealing.
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Additionally, to ensure more reliable results, we selected a field of cells and took photographs of the fluorescence channel, bright field channel, and phase contrast channel separately. By comparing the three images, it can be observed that the GFP fluorescence is located in living cells. This confirms that the observed GFP fluorescence is the result of cellular protein expression, rather than fluorescence caused by errors such as cellular debris.
