Team:SCUT-China/Experiments

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Project

1.PDE5A Silencing Device

Vector Map

The vector we used in our experiment was created using scarless golden gate assembly. We cloned the silencing device into a psb1c3 vector after our experiment.

Fig.1 Lentivirus vector cointaing PDE5a silencing device

In our experiment we designed three PDE5A silencing devices and chose the best one. Before our experiment we made a preliminary experiment. We used 10umol/L sodium nitroprussiate(SNP),a NO donor, to treat HEK 293 cells for different time gradient as the positive control . The results are as followed

Fig.2 cGMP concentration after treated with 10umol/L SNP

We used RT-PCR method to see whether PDE5a gene was silenced by our shRNA.

Fig.3 PED5a transcription level after transfection with three different shRNA

From the results of Fig.2 we could see that PDE5a was almost silenced by all the three different shRNA. So we could not decide which device was the best one .So we used Elisa Kit to detect cGMP concentration and used the results to select the best silencing device.

Fig.4 cGMP concentration after treated with three different shRNA

From the results of Fig.3 we choose shRNA3 (part number: BBa_K1720005)as the best PDE5A silencing device.

2.sGC Overexpression

Vector Map of Alpha3 subunit

Fig.5 Lentivirus vector cointaing guanylate cyclase 1 alpha3 subunit gene

Vector Map of Bata3 subunit

Fig.6 Lentivirus vector cointaing guanylate cyclase 1 beta3 subunit gene

HEK293 cells were transfected by designed vectors. Vectors carrying alpha3 subunit of sGC was inserted mCherry gene as a reporter while vectors carrying beta3 subunit of sGC was inserted EGFP gene as a reporter. Both red fluorescence signal and green fluorescence can be observed under fluorescence microscope. It indicated that the transfection was successful.

Fig.7 Red fluorescence signal after transfection of alpha3 subunit

Fig.8 Green fluorescence signal after transfection of beta3 subunit

sGC alpha3 subunit and beta3 subunit gene expression levels were determined by real-time PCR

Fig.9 Transcription level after transfection with alpha3 and beta3 subunit

Fig.10   △CT vs GAPDH after transfection of alpha3 and beta3 subunit

As we up-regulate the transcriptional level of sGC alpha3 subunit and bata3 subunit, we used sGC Elisa Kit to detect the sGC activity to see whether two subunits combine with each other successfully.

Fig.11 sGC activity after transfection of sGC vectors and shRNA vectors

We used Elisa kit to detect the sGC activity. From the result we can see that the activity of sGC will be improved after overexpression but if we silence PDE5A gene the activity of sGC will be up regulated rapidly.

We used cGMP Elisa kit to detect the cGMP level to see whether cGMP concentration will be up regulated by overexpression of sGC.

Fig.12 cGMP concentration after transfection of sGC vectors and shRNA vectors

From the results (Fig.8 and Fig9)we can see that once the activity of sGC is up regulated the cGMP concentration will be up regulated simultaneously.

3.Hypoxia Responsive Promotor

Vector Map

Fig.13 Vecror containing hypoxia responsive CMV promotor and EGFP reporter

We designed a hypoxia responsive CMV promotor by inserting a hypoxia responsive element （HRE） to CMV promotor. We transiently transfected HEK293 cells with plasmids containing hypoxia-induced promotor and EGFP reporter. The positive control was transiently transfected with plasmids that contain CMV promoter and EGFP reporter. The control group was transiently transfected with plasmids that contain hypoxia responsive promotor and culture under aerobic situation. The experimental group group was transiently transfected with plasmids that contain hypoxia responsive promotor and the cells were treated with sodium hyposulfite, an oxygen cleaner to cause hypoxia situation, for 2 hours.

Fig.14 EGFP signal under the control of CMV promotor

Fig.15 EGFP signal under the regulate of HRE in aerobic situation

Fig.16 EGFP signal under the regulate of HRE in hypoxia situation

From the results above, we can see that hypoxia responsive promotor still working under aerobic situation. So that this promotor is not a strict hypoxia responsive promotor as we expect.