Team:Tongji Shanghai/Plasmid Construction
Overview
- Cancer thermotherapy realized by depositing heat into tumor in a minimally invasive way is a promising alternative to the conventional therapies for cancer treatment. It’s a therapeutic tool to eradicate cancer tumor with minimum toxic effects. To make it better, we plan to improve its ability to target to tumor cells and thermosensitivity of tumor cells. Furthermore, we hope it can reflect the the treatment process. Here, we provide an approach to optimize its tumor targeting and thermosensitivity of tumor cells by hTert promoter and heat shock protein 70(hsp70) prompter and the following tumor suppressor p53. Additionally, the luciferase following p53 makes it a reporter system.
Figure1
Experiment:
- Now let’s see the design of our experiments and how we did them.
1.Hsp70-p53-luciferase part.
- To test if the plasmid is usable, we transfected it into cells to see if the expression of p53 would increase with heat treatment. Furthermore, the transfected cells without heat treatment were passaged to 96 well plate, increasing the temperature by laser so we could test the survival of the cell.
The plasmid was transfected by PEI, heat shock of cells was performed in 42C water bath.
2.HTert-p53 part.
- To test if the hTert promoter works, we construct a plasmid in which the promoter is linked with GFP. By observing the cell in fluorescence microscope and comparing the expression of
GFP in the cell transfected the plasmid and cell without tert, we could make sure p53 following the promoter can overexpress in tumor cells specifically. Next, over expressed hTert-p53 in breast cancer cell line hcc 1937 and treated it with laser then analyzed the survival rate of cell.
Result:
Hsp70-p53-luciferase part：
1.The result of transfection
- Co-transfected GFP for the data analysis of laser experiment could be obviously seen from the cell.
2.Fluorescence-activated cell sorter analysis
- To make the analysis of cell survival rate more accurate, a GFP plasmid was cotransfected into the cell, we could get the efficiency of transfection by FACS.
3.Western blot
- Cell transfected with Hsp70-p53-luciferase and GFP was heat shock by 42C water bath, after restoration in 37C for one hour, protein was extracted to do western blot showing the expression of p53.
Hsp70-p53-luciferase part：
1.GFP expression
- HTert-GFP was overexpressed in both breast cancer cell hcc 1937 and test negative cells. Observed the cell in fluorescent microscope directly so we could see that the hTert prompter is able to make the gene after it transcripted in tumor cells specifically.
2.Cell survival rate
- After hTert-p53 was overexpressed in tumor cell, it was passaged into 96 well plate to accept laser treatment so we could analyze its survival rate.
- The result is shown in Cell survival part.
