Difference between revisions of "Team:Tongji Shanghai/Plasmid Construction"

Overview

Cancer thermotherapy works as depositing heat in tumor resulting minimal damage, which is a promising alternative to the conventional therapies for cancer treatment. The advanta-ge of it is no toxicity to normal cells because of physical treatment. To make it better, we plan to improve its ability by genetically engineering, focusing on tumor cell’s targeting and tumor cell’s thermo-sensitivity. Furthermore, we hope to reflect the treatment process. Here, we provide an approach to optimize its targeting and thermo-sensitivity by hTERT promoter and heat shock protein 70(hsp70) prompter and the following tumor suppressor p53. Additionally, the luciferase following p53 turn it into a reporter system.

Now let’s see the design of our kit and how we achieve them.

1.Hsp70-p53-luciferase part.

To test if the plasmid is working, we transfected it into cells to see if the expression of p53 increases with heat treatment. Furthermore, the trans-fected cells without heat treatment were removed to 96 well plate, in-creasing the temperature so we could test the cellular viability.

The plasmid was transfected by PEI, heat shock of cells was performed in 42°C incubator

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 compare GFP expression in both telomerase possitive and negative cells, we could make sure p53 following the promoter can over express in tumor cells specifically. Next, we infected hTERT-p53 into breast cancer cell line hcc 1937 and treated it with 42°C then ana-lyzed the cellular viability.

RESULTS

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

The transfection efficiency in 293T cell line.

The transfection efficiency in hcc 1937 cell line.

To make the analysis of cell survival rate more accurate, a GFP plasmid was co-transfected into the cell, we could get the efficiency of transfection by FACS.

Western blot

Our part require hTERT promoter to reach specifically targeting, so we have designed experiment to illustrate that the part works as we expected.

hTERT-GFP was overexpressed in both breast cancer cell hcc 1937 and telomerase-negative cells. Observed the cell in fluorescent microscope directly so we could see that the hTERT prompter is able to make the downstream gene transcripted in tumor cells specifically.

Cellular viability

After hTERT-p53 was over expressed in tumor cell, it was removed into 96-well plate to accept la-ser treatment so we could analyze the cellular viability.

The result is shown in Cell experiment part.

Biosafety: