iGEM 2022 — Team subcat-shanghai

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{% block title %}Notebook{% endblock %}
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Notebook

0508

1. T98G and U118 cells recovery and culture in DMEM
2. DMEM (FBS) cell culture medium preparation

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook01.jpg)

0510-0524

1. transfer the T98G and U118 cells into the fresh DMEM medium containing 50μM and 200μM TMZ, respectively.
2. The culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for
two weeks.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook02.jpg)

0525-0608

1. Transfer the T98G and U118 cells incubate in TMZ-containing medium into the fresh DMEM medium containing 100μM
and 400μM TMZ, respectively.
2. The culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for
two weeks.
3. screen for TMZ-resistance-related genes in a bioinformatics way

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook03.jpg)

0609- 0623

1. transfer the T98G and U118 cells into the fresh DMEM medium containing 200μM and 800μM TMZ, respectively.
2. The culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for
two weeks.
3. designed the siRNA of PDRG1 gene plasmids and send them to the company for plasmids construction and lentivirus
package.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook04.jpg)

0624-0707

1. transfer the T98G and U118 cells into the fresh DMEM medium containing 400μM and 1600μM TMZ, respectively.
2. The culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for
two weeks.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook05.jpg)

0708-0721

1. transfer the T98G and U118 cells into the fresh DMEM medium containing 800μM and 3200μM TMZ, respectively.
2. The culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for
two weeks.
3. After the successful preparation of TMZ-resistant cells, the culture is maintained at a low concentration of TMZ
(different cell concentrations vary).
4. transfer the cells to a 6-well plate

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook06.jpg)

0722-0723

1. transfect the TMZ resistance T98G and U118 cells with lentivirus of PDRG1-siRNA
2. change the culture medium the next day and screened them using purine
3. measure the resistance of TMZ of the transfected cell line

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook07.jpg)

0724

1. collect the transfect cells and detect the green fluorescence
2. total RNA extraction of the transfected cells
3. measure the abundance of PDRG1 gene transcripts by qPCR

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook08.jpg)

0725

1. measure the expression level of PDRG1 protein by Western blot

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook09.jpg)
![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-notebook10.jpg)

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{% block title %}Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-banner.jpg)
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Engineering

Introduction

Glioma, the most common malignant tumor of the adult central nervous system, has a high mortality rate. The
combination of temozolomide (TMZ) chemotherapy and radiotherapy is the current standard for curing gliomas, but most
patients eventually develop resistance to TMZ. To better individualize and precision therapy, finding new molecular
targets that increase TM therapeutic sensitivity is really important.

In our project, we screened out the gene PDGR1 (p53 and DNA damage regulated 1) which is related to TMZ resistance
through bioinformatics analysis. To verify its role in the TMZ resistance process, we developed a U118
TMZ-resistance cell line and knocked down the PDRG1 gene by siRNA. Then we measured the transfected cell line’s
lifespan in different concentrations of TMZ. Our experimental data indicated that PDRG1 could be a target gene to
surmount TMZ resistance.

How we construct the TMZ-resistance cell line

We chose the U118 cell line as the Initial cell line to carry out the project. The drug concentration of the
starting TMZ is set with each cell line IC50, the culture medium containing TMZ is changed every two days, and the
same concentration of TMZ is maintained for two weeks. Then calculate the new IC50, increase the TMZ concentration,
and TMZ is maintained for two weeks, using the same method to change the culture medium. Repeat this until IC50
increases to 500 μM.

After the successful preparation of TMZ-resistant cells, the culture is maintained at a low concentration of TMZ
(different cell concentrations vary). The control cells were treated with the same concentration of DMSO, and the
processing time and procedure were the same plus TMZ. The cell line is established and expanded, including breed
conservation.

The CCK8 cell viability assay was used to examine the TMZ tolerance of the cells. Both the control group
(transfected with NC plasmid) and the experimental group (TMZ-resistance cell line) of U118 cell lines were tested.
Raw data from the cell viability assay was normalized and listed below in Table 1.

Table 1. The left column lists different shRNA concentrations, varying from 0-3200 μM. U118 represents the control
group. U118-R are the experimental groups.

|  | U118 | | | U118-R | | |
| --- | --- | --- | --- | --- | --- | --- |
| 0 μM | 104.924 | 95.81989 | 99.25614 | 102.8273 | 102.9316 | 94.24102 |
| 200 μM | 88.38738 | 80.564 | 81.64222 | 97.39861 | 89.6976 | 90.34763 |
| 400 μM | 78.38556 | 70.02831 | 65.39635 | 91.79606 | 86.28042 | 81.37891 |
| 800 μM | 37.89191 | 53.59028 | 45.89368 | 87.6825 | 73.69641 | 78.31982 |
| 1600 μM | 22.42354 | 15.71259 | 12.30861 | 70.20858 | 65.61993 | 75.28621 |
| 3200 μM | 0.243256 | 0.71806 | 1.35164 | 65.33604 | 53.77752 | 57.18424 |

We can observe that TMZ-treated cells have significantly higher viability than the control group (Figure 1). This
illustrates that we successfully screened U118 TMZ-tolerance cell lines.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-engineering01.png)
Figure 1. The result of CCK8 assay for U118 cell line. The black line represents the data from the control
group transfected with NC plasmid. The red line represents the TMZ-resistance cell line we screened. T-test was
done to measure the significant difference between the control and experimental group start to show up (ns:
P>0.05, \*: P≤0.05, \*\*: P≤0.01, \*\*\*: P≤0.001).

How we test PDRG1

a) U118 PDRG1 gene knockdown cell line construction

In order to verify if knockdown of the PDRG1 gene could decrease the TMZ-resistance
cells’ lifespan, we transfected U118 cells with Lentivirus packaged plasmids containing siRNAs and screened them
using purine. The fluorescence microscope can be used to check if the plasmids have been transfected successfully by
detecting the green fluorescence (Figure 2).

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-engineering02.png)
Figure 2. Detection of green fluorescent protein expression in the stable knockdown cell using fluorescence
microscopy. The green fluorescence proves successful plasmid transfection.

b) Measure the mRNA abundance and protein expression level of PDRG1 knockdown U118 cell line

To get more reliable and quantitative results, fluorescence real-time quantitative PCR was done to test the mRNA
expression of PDRG1 in the transfection cell line (Figure 3). As shown in figure 3, the abundance of the PDRG1 gene
in the transfected cell line, the siRNA successfully inhibited the PDRG1 transcription.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-engineering03.png)

Figure 3. the result of mRNA abundance of the control group and PDRG-1 knockdown group

In addition, the Western blot was done to further confirm PDRG-1 expression on the
protein level in each group. The thickness of the protein band indicates the amount of the expressed protein. GAPDH
was used as a control group, as it is a common housekeeper gene. It can be easily observed that in the PDRG-1 group,
the protein band of Lv-sh1 and Lv-sh2 are thinner than the Lv-shNC, showing the lowered expression of PDRG-1 in the
experimental group (Figure 4). In addition, the Western blot was done to further confirm PDRG-1 expression on the
protein level in each group. The thickness of the protein band indicates the amount of the expressed protein. GAPDH
was used as a control group, as it is a common housekeeper gene. It can be easily observed that in the PDRG-1 group,
the protein band of Lv-sh1 and Lv-sh2 are thinner than the Lv-shNC, showing the lowered expression of PDRG-1 in the
experimental group (Figure 4).

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-engineering04.png)
Figure 4. Western blot result of PDRG-1 and GAPDH.

c) TMZ-tolerance of the U118 PDRG1 knockdown cell line

The control group and the PDRG-1 knockdown group are treated with TMZ. The relative cell
viability was recorded over 72 hours for U118 (Figure 5). In both groups, there are significant differences between
the control group and the PDRG-1 knockdown group. Comparatively, the PDRG-1 knockdown groups show lower cell
viability over time and lower TMZ tolerance, which is our expected result.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-engineering05.png)

Figure 5. Measure the resistance of PDRG1 knockdown U118 cell line and the control cell line

How we learn from our project

We have already collected the figures from our experiments. PDRG1 is a small oncogenic protein related to TMZ
resistance. However, TMZ resistance may cause chemotherapy failure which significantly decreases the survival rates
of GBM patients. To conquer this problem, we screened the databases to search for related genes that could be used
as therapeutic targets, so PDRG1 was chosen for our project.

We constructed a PDRG1 knockdown cell line that has been shown to have a higher potential to counter TMZ tolerance.
This cell line can be used to further investigate the PDRG signaling pathway to gain a deeper understanding of the
molecular mechanisms of TMZ tolerance development and thereby inspire new research directions seeking to eliminate
or moderate TMZ tolerance. We believe that if we can fully understand the mechanism of PDRG1 in the TMZ-resistance
process, we could provide new ways to treat GBM.

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Experiments

1. Plasmid Extraction

1.1. Luria-Bertani Broth preparation

1.1.1. Weigh 10g tryptone, 5g yeast extract, and 10g sodium chloride (2:1:2)
1.1.2. Mix the tryptone, yeast extract, and sodium chloride with double distilled water to 1L in total
1.1.3. Use the magnetic stirrer to mix the mixture thoroughly
1.1.4. Send the prepared LB broth to the autoclave for sterilization

1.2. Culturing the Bacteria

1.2.1. Add 250μl ampicillin solution into 25ml sterilized LB medium
1.2.2. Add 10μl Escherichia. coli solution into 7ml sterilized LB medium
1.2.3. Incubate the bacteria in LB medium in the shaker overnight (220rpm in 37 °C)

1.3. Plasmid Extraction

1.3.1. Dispense the overnight cultured bacteria into 2ml EP tubes, 1.7ml per tube.
1.3.2. Centrifugate of the bacterial solution (12000rpm for 2min at room temperature)
1.3.3. Remove the supernatant
1.3.4. Add 250μl Buffer SP1 and disperse the precipitated bacteria with a pipette
1.3.5. Add 250μl Buffer SP2 and mildly flip the EP tube 5-10 times then rest for 2-4min
1.3.6. Add 350μl Buffer SP3 and mildly flip the EP tube 5-10 times then centrifugate the solution (12000rpm for
10min)
1.3.7. Place the adsorption column into a new EP tube; then add 500μl Buffer S and centrifuge 12000rpm for 5min in
order to balance the environment of the adsorption column
1.3.8. Transfer the supernatant from 1.3.6 into the adsorption column then centrifugate (12000rpm for 5min)
1.3.9. Remove the filtrate at the bottom of the EP tube
1.3.10. Add 500μl Buffer DW1 into the adsorption column then centrifugate (12000rpm for 1min) and repeat
1.3.9
1.3.11. Add 500μl wash solution (with anhydrous ethanol) into the adsorption column then centrifugate (12000rpm
for
1min) and repeat 1.3.9
1.3.12. Repeat 1.3.11
1.3.13. Transfer the adsorption column into a new EP tube then centrifugate (12000rpm for 2min)
1.3.14. Add 50-100μl Elution Buffer on the adsorption membrane and rest for 1-2min then centrifugate (12000rpm for
2min)
1.3.15. Take 1μl product and measure the plasmid concentration by spectrophotometer

2. Cell Resuscitation

2.1.1. Mix 45ml Dulbecco’s Minimal Essential Medium (DMEM) and 5ml Fetal Bovine Serum (FBS) (9:1)
2.1.2. Take out the cells in the cryopreservation refrigerator and melt them in 37°C water
2.1.3. Add 500μl cells into the 5ml growth medium and dispense to 3 EP tubes
2.1.4. Centrifugate the cells (1000rpm for 3min at room temperature)
2.1.5. Remove the supernatant
2.1.6. Disperse the precipitated cells by pipette with 0.5ml growth medium
2.1.7. Pour 4ml growth medium into Petri dishes and inoculate cells
2.1.1. Mix 45ml Dulbecco’s Minimal Essential Medium (DMEM) and 5ml Fetal Bovine Serum (FBS) (9:1)
2.1.2. Take out the cells in the cryopreservation refrigerator and melt them in 37°C water
2.1.3. Add 500μl cells into the 5ml growth medium and dispense to 3 EP tubes
2.1.4. Centrifugate the cells (1000rpm for 3min at room temperature)
2.1.5. Remove the supernatant
2.1.6. Disperse the precipitated cells by pipette with 0.5ml growth medium
2.1.7. Pour 4ml growth medium into Petri dishes and inoculate cells

3. RNA Level Examination

3.1. RNA Extraction

3.1.1. Remove the growth medium of the Petri dishes
3.1.2. Use 0.5ml Trizol and resuspend the cells and collect the cells into EP tubes
3.1.3. Rest the cells after lysed for 5mins under room temperature
3.1.4. Add 0.2ml chloroform into the EP tube and shake drastically then centrifugate (12000rpm for 10min)
3.1.5. Transfer 200μl upper aqueous phase (RNA) to new EP tube, then add 100μl anhydrous ethanol and blend
completely
3.1.6. Put the adsorption column into the new EP tubes then add the solution from 3.1.5 on the adsorption membrane
(12000rom for 30s)
3.1.7. Add 500μl RPE solution to the adsorption membrane and rest for 2min then centrifugate (12000rpm for 30s)

3.1.8. Repeat 3.1.7
3.1.9. Centrifugate the empty EP tube in 10000rpm for 2min
3.1.10. Add 30μl DEPC-treated ddH2O on the central of the adsorption membrane and rest for 5min then centrifugate
(12000rpm for 2min)
3.1.11. Repeat 3.1.10

3.2. RNA Reverse Transcription

3.2.1. Add 1μl dNTPs Mix and 1μl Oligo-dT Primer to 2μl template RNA; then add RNase-free water up to 10μl
3.2.2. Put the template into the PCR amplifier with setting 65°C for 5min then rest at 4°C
3.2.3. Add 4μl 5× RTase Reaction Buffer, 0.5μl RNase Inhibitor, and 0.5μl Evo M-MLV-RTase into the 10μl annealed
template then add the RNase-free water up to 30μl
3.2.4. Put the template into the PCR amplifier with setting 42°C for 15-30min then 95°C for 5min and rest at
4°C
3.2.5. Add 7.5μl TB green, 0.3μl Rox, 0.3 μl forward primer, 0.3 μl reverse primer and 4.6 μl ddH2O with 2μl cDNA
template
3.2.6. Centrifugate the 96-well plate (1000rcf for 3min)
3.2.7. Proceed qPCR

4. Protein Level Examination

4.1. Protein Extraction

4.1.1. Cell lysis Buffer was prepared according to 5X Buffer, 5× NaCl, 5× SDS, 5× DOC, and 5× NP-40.
4.1.2. Centrifuge at 700 ×g for 5 min and discard the supernatant
4.1.3. Wash the cells twice by resuspension in PBS then centrifuged at 4°C and remove the supernatant
4.1.4. Resuspended the precipitate in cell lysis buffer, immersed in an ice pack, and incubated on a shaker for
30min
4.1.5. Centrifuged the lysed cells at 12000 ×g for 10min at 4°C to precipitate cell debris
4.1.6. Transfer protein supernatant to EP tube and store at -80°C

4.2. SDS-PAGE Gel Preparation

4.2.1. Add the 3.7ml separation gel solution and separation gel buffer with 60μl modified coagulant
4.2.2. Pour a certain amount of solution into the plate to make the distance between the fluid level and the top
edge of plate is 0.5cm longer than the comb
4.2.3. Pour ddH2O in the plate in order to compact the separation gel
4.2.4. Remove the water after the separation gel is solidified
4.2.5. Mix 0.75ml stacking gel solution and stacking gel buffer with 15μl modified coagulant
4.2.6. Pour the solution into the plate and then insert the comb
4.2.7. Leave it for about 15min for consolidation

4.3. Gel electrophoresis

4.3.1. Install the gel in the Electrophoresis and soak in the SDS-PAGE Running Buffer
4.3.2. Add 20μl protein template into the hole on the gel
4.3.3. Set the Electrophoresis for 90V

4.4. Western Blot

4.4.1. Add one pack of transfer solution and 200ml methanol then add ddH2O up to 1000ml
4.4.2. Cut the filter paper and membrane
4.4.3. Soak the filter paper, membrane, and gel in the transfer buffer
4.4.4. From the bottom to top: stent, sponge, 3 filter paper, gel, NC member, 3 filter paper, sponge, stent
4.4.5. Place the sandwich into the transfer room for 1h
4.4.6. Mix 5ml 20× TBS with 90ml ddH2O and 1 pack of blocking reagent to make the blocking solution
4.4.7. Soak the NC membrane into Ponceau S 10min
4.4.8. Soak the NC membrane into the blocking solution and place it on the shaker for 1h
4.4.9. Pour the blocking solution out and add new blocking solution into the box then add primary antibody of
PDRG-1 to larger hole membrane, GAPDH to smaller hole membrane; culturing on the shaker in 4°C overnight
4.4.10. Add 10ml 20× PBST into 190ml ddH2O, then add 100μl Tween-80
4.4.11. Pour the primary antibody out, then add the mixture from 4.4.10 and shake for 3 times per 5min
4.4.12. Use TBS wash the NC membrane and soak it in the blocking solution then add the HRP secondary antibody and
place it on the shaker for 1h
4.4.13. Pour out the blocking solution and wash the NC membrane by TBST on the shaker for 3 times per 5min; then
wash it by TBS again
4.4.14. Prepare the DAB solution by adding 1 pack of DAB, 1ml 10× DAB development solution, 9ml ddH2O, and 10μl
hydrogen peroxide
4.4.15. Place the NC membrane with adding developer in the Invitrogen E-Gel Imager

5. Screen for target genes in a bioinformatic way

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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Results

1. Glioma cell line TMZ resistance verification

T98G and U118 cells were selected. The drug concentration of the starting TMZ is set with each cell line IC50, the
culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for two
weeks. Then calculate the new IC50, increase the TMZ concentration, and TMZ is maintained for two weeks, using the
same method to change the culture medium. Repeat this until IC50 increases to 500 μM. After the successful
preparation of TMZ-resistant cells, the culture is maintained at a low concentration of TMZ (different cell
concentrations vary). The control cells were treated with the same concentration of DMSO, and the processing time
and procedure were the same plus TMZ. The cell line is established and expanded, including breed conservation.
The CCK8 cell viability assay was used to examine the TMZ tolerance of the cells. Both the control group
(transfected with NC plasmid) and the experimental group (TMZ-resistance cell line) of T98G and U118 cell lines were
tested. Raw data from the cell viability assay was normalized and listed below in Table 1.

Table 1. The left column lists represent different shRNA concentrations,
varying from
0-800 μM. T98G and U118
represent the control group. T98G-R and U118-R are the experimental groups.

|  | T98G | | | T98G-R | | |
| --- | --- | --- | --- | --- | --- | --- |
| 0 μM | 99.71424 | 95.15717 | 105.1286 | 106.2187 | 88.18571 | 105.5956 |
| 50 μM | 93.75846 | 84.68943 | 94.14198 | 92.83445 | 98.89799 | 94.46365 |
| 100 μM | 61.81381 | 56.39495 | 53.963 | 85.97434 | 90.26267 | 86.27367 |
| 200 μM | 40.81757 | 50.4142 | 53.10573 | 86.81124 | 72.16249 | 74.94807 |
| 400 μM | 26.39495 | 31.26786 | 20.84524 | 88.23946 | 69.41845 | 79.48076 |
| 800 μM | 14.13716 | 9.741315 | 6.88946 | 62.37019 | 66.14539 | 74.58155 |

|  | U118 | | | U118-R | | |
| --- | --- | --- | --- | --- | --- | --- |
| 0 μM | 104.924 | 95.81989 | 99.25614 | 102.8273 | 102.9316 | 94.24102 |
| 200 μM | 88.38738 | 80.564 | 81.64222 | 97.39861 | 89.6976 | 90.34763 |
| 400 μM | 78.38556 | 70.02831 | 65.39635 | 91.79606 | 86.28042 | 81.37891 |
| 800 μM | 37.89191 | 53.59028 | 45.89368 | 87.6825 | 73.69641 | 78.31982 |
| 1600 μM | 22.42354 | 15.71259 | 12.30861 | 70.20858 | 65.61993 | 75.28621 |
| 3200 μM | 0.243256 | 0.71806 | 1.35164 | 65.33604 | 53.77752 | 57.18424 |

We can observe that TMZ-treated cells have significantly higher viability than the control group (Figure 1). This
illustrates that we successfully screened TMZ-tolerance cell lines.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-01.png)
Figure 1. The line charts of CCK8 assay for A) U118 and B) T98G cell line. The black line represents the data
from the control group transfected with NC plasmid. The red line represents the TMZ-resistance cell line we
screened. T-test was done to measure the significant difference between the control and experimental group start
to show up (ns: P>0.05, \*: P≤0.05, \*\*: P≤0.01, \*\*\*: P≤0.001).

2. Screen for target genes in a bioinformatic way

Through extensive literature review, we found that PDRG1, the down-regulated differential gene, was up-regulated in
various tumor tissues, such as colorectal cancer, ovarian cancer, lung cancer, breast cancer, and endometrial
cancer. At the same time, PDRG1 down-regulation can enhance the radiosensitivity of nasopharyngeal cancer (Figure
2A).
Therefore, PDRG1 was preliminarily identified as a potential target gene, and the function of PDRG1 was verified by
using glioma-related data from TCGA and GTEx databases. Firstly, the expression of PDRG1 in glioma tumor samples and
normal samples was analyzed, and the expression of PDGR1 in normal samples was lower than that in tumor samples.
Secondly, whether PDRG1 affects the prognosis of glioma was analyzed, and patients with low expression of PDRG1 had
a better prognosis and longer survival time (Figure 2B). What’s more, we observed that the expression of PDRG1 in
GBM and LGG was lower in normal samples than in tumor samples. And in GBM and LGG, the survival time of samples with
low PDRG1 expression was significantly higher than that of samples with high PDRG1 expression (Figure 2C). Both OS
and RFS can reach the same conclusion. So again, it verifies our hypothesis. In conclusion, PDRG1 was taken as the
target gene for further verification by cell assay.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-02.jpg)
Figure 2. PDRG1 plays an important role in tumors. A. the expression level of PDRG1 in different kinds of
cancer, B. the survival time of patients with the different expression levels of PDRG1, C. the survival time of
GBM and LGG patients with the different expression levels of PDRG1.

3. PDRG-1 knockdown cell-line construction

In order to verify if knockdown of the PDRG1 gene could decrease the TMZ-resistance
cells’ lifespan, we transfected T98G cells and U118 cells with Lentivirus packaged plasmids containing siRNAs and
screened them using purine. The fluorescence microscope can be used to check if the plasmids have been transfected
successfully by detecting the green fluorescence (Figure 3).

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-03.png)

Figure 3. Detection of green fluorescent protein expression in the stable knockdown cell using fluorescence
microscopy. The green fluorescence proves successful plasmid transfection.

To get more reliable and quantitative results, fluorescence real-time quantitative PCR
was done to test the mRNA expression of PDRG1 in the transfection cell line (Figure 4). As shown below, the mRNA
expression level of PDRG-1 is significantly lower in shRNA treated group (Table 2).

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-04.png)
Figure 4. Bar graph of mRNA level of the control group and PDRG-1 knockdown group.

Table 2. Data of relative expression of PDRG-1 from qPCR.
![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-05.jpg)

4. PDRG-1 knockdown cell line PDRG1 protein expression level measurement

In addition, the Western blot was done to further confirm PDRG-1 expression on the
protein level in each group. The thickness of the protein band indicates the amount of the expressed protein. GAPDH
was used as a control group, as it is a common housekeeper gene. It can be easily observed that in the PDRG-1 group,
the protein band of Lv-sh1 and Lv-sh2 are thinner than the Lv-shNC, showing the lowered expression of PDRG-1 in the
experimental group (Figure 5).

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-06.png)
Figure 5. Western blot result of PDRG-1 and GAPDH.

5. TMZ tolerance test 5. TMZ tolerance test

The control group and the PDRG-1 knockdown group are treated with TMZ. The relative cell viability was recorded over
72 hours for U118 (Figure 6A) as well as T98G (Figure 6B).
In both groups, there are significant differences between the control group and the PDRG-1 knockdown group.
Comparatively, the PDRG-1 knockdown groups show lower cell viability over time and lower TMZ tolerance, which is our
expected result.

![](https://static.igem.wiki/teams/4251/wiki/t-subcat-shanghai-result-07.png)
Figure 6. TMZ tolerance of different glioma cell lines over time. The significance is indicated near the data
point using the same abbreviation as listed above. A. the result of the U118 cell line, B. the result of the T98G
cell line

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