Team:NAU-CHINA/testh

Fig.5. Function verification of recombinases in HEK 293T cell Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase recognition sites and corresponding recombinase gene. The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (the first column picture) or transfected with plasmids containing corresponding combination of promoters and recombinase genes (other column pictures) together, are shown.

The results show that the recombinases can recognise the sites and reverse the sequence between sites in HEK 293T. 

Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells

Fig.6. Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells HEK 293T cells were co-transfected with six different amounts of plasmids containing recombinase genes (miniCMV-Bxb1 and miniCMV-TP901) , and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co-transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. Transfection of different amounts of plasmids containing recombinase genes into cells indicates that cells can produce recombinase at different concentrations. The experiment was repeated three times. (A) Fluorescence microscope observation of HEK 293T undergone different experimental treatments (B) The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. (C) The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions. The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.