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PDRG1-SiRNA-1

RDRG1-SiRNA-1

Profile

Name: PRDG1-siRNA Base Pairs: 21bp Origin: Company synthetic Properties: Silencing RNA of PDRG1

Contribution

siRNA is a double-stranded non-coding RNA molecule, it usually between 20 and 24 base pairs, and it is also known as silencing RNA and short interfering RNA. siRNAs have very tight target specificity, this anti-sense strand binds to the target mRNA, and then the target mRNA cleavage is induced. PRDG1-siRNA is designed according to the DNA sequence of PRDG1, and it’s 21bp long. We insert the siRNA into AgeI and EcoRI sites of the GV248 plasmid, and the correct plasmid was sent to the company for lentivirus packaging. The lentivirus was then transfected to T98G and U118 TMZ resistance cell line to knock down the PRDG1 transcripts.

Engineering Success

1. 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 1).

Figure 1. 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 2). As shown below, the mRNA expression level of PDRG-1 is significantly lower in shRNA treated group (Table 2).

Figure 2. Bar graph of mRNA level of the control group and PDRG-1 knockdown group.

Table 1. Data of relative expression of PDRG-1 from qPCR.

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 3).

Figure 3. Western blot result of PDRG-1 and GAPDH.

Proof of function

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 4A) as well as T98G (Figure 4B). 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.

Figure 4. 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

Figure 4. 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

Sequence and Features