EAAT2-Functional Characterization
At the beginning of our project, we characterized the function of EAAT2 and its expression, and simulated the inflammation-inducing environment for Neuro-2a cells using the high-glutamate condition exhibited in neurodegenerative patients’ cerebrospinal fluid.
In this part of test, we focused on two impacts our exosome medicine may bring to the patients:
Reducing the excitatory amino acids in the cerebrospinal fluid.
Alleviate the cytotoxicity brought by excessive glutamate or the excitotoxicity induced.
1. Glutamate Assay
We tested two sets of cells: the EAAT2-transfected N2a cells and the empty plasmid pcDNA3.1-transfected cells (negative control). For each set of N2a cells, the time interval for changing the DMEM medium to inflammation-inducing medium was three hours. There were four groups (approximately 0 hour, 3 hours, 6 hours and 9 hours) in total which were the time to change the medium. The level of glutamate in the culture medium was then measured using the glutamate assay kit and the absorbance for each sample was read in a plate reader.
The distribution of each sample are displayed as below:
Figure 1. The expression of EAAT2 helps reduce the glutamate level in cell medium. The glutamate concentration in cell medium grows significantly faster in EAAT2-untransfected groups, which indicates that the expression of EAAT2 can help hold the concentration of extracellular glutamate.
Figure 2. The glutamate concentration in pcDNA3.1(+)-transfected cell medium is significantly higher than those of EAAT2-transfected groups after six hours, Student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).
The results of glutamate assay reveals that the expression of EAAT2 on cell membrane maintained the concentration of glutamate in a relatively stable level, and significantly reduced the concentration of glutamate in the cell medium. Higher the glutamate concentration is, the more significant EAAT2 removed the glutamate in cells. In addition, the expression of EAAT2 did not over-transport glutamate and maintained the glutamate concentration similar to the origin level.
2. WST-1 Cytotoxicity Test
To detect the cytotoxicity induced by the excessively high glutamate concentration, we used the WST-1 assay as an alternative to the MTT assay for our test. The dehydrogenase in mitochondria can reduce the the tetrazolium salt WST-1 to soluble and chromatic formazan, and a high absorbance measured indicates a low cytotoxicity induced by glutamate. The absorbance of these samples were measured at 450 nm, and 690 nm was used as a reference wavelength.
The distribution of each sample are displayed as below:
Figure 3. The cytotoxicity induced by high concentration of glutamate (in EAAT2-transfected cell medium) can be obviously alleviated as the A450-A690 is remarkably lower than that of pcDNA3.1(+)-transfected cells.
Figure 4. The comparison in A450- A690 between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells (after a two-hour incubation in 37 Celsius), Students' t-test was applied (*: p<0.05, **: p<0.01, ***: p<0.001).
The results of WST-1 indicated that the expression of EAAT2 on cell membrane could significantly alleviate the excitotoxicity induced by glutamate. The cytotoxicity may have limited influence on cell proliferation even if the cells are cultured in inflammation-inducing (80 uM glutamate) for longer than 9 hours.
C/D Box-L7Ae-Functional Characterization
In this part of experiments, we investigated the function of L7Ae, which may binds the C/D Box on our designed mRNA, and anchor the therapeutic mRNA on the inner face of exosomes.
The wet lab focused on two impacts L7Ae may bring to the exosome medicine:
The production of exosome in exosome donor (HEK293T cells)
The RNA anchored in the exosome
1. Nanoparticle Tracking Assay
After the engreen co-transfection of L7Ae with C/D Box-nluc/nluc/empty pcDNA3.1(+), we conducted NTA to detect if the transfection may influence the exosome production, and ensure that the concentration reached 2x105 /ul, which is the recommended exosome concentration in exosome transfection (Exo-fect) in the sample ultracentrifugated. We distributed the transfected HEK293T cells in a 12-well plate as below:
We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:
Result1
Result2
Result3
The original concentration of particles in three groups were all measured to be 1.3x1010 particles/ml, which is approximately 50x recommended concentration for exosomes to be transfected by Exo-fect.
For the particles with different diameters, the distribution of their concentration in each set of samples are listed as below:
Figure 5. The numbers of ~150-nm-diameter particles with different diameters measured by NTA in a 50 ul ultracentrifuged exosome solution were almost the same (of cells those transfected with (1) nluc+L7Ae (2) pcDNA3.1(+)+L7Ae (3) C/D Box-nluc+L7Ae), the solution was diluted with a factor of 300x.
These results shows that the co-transfection and expression of the plasmids listed would not affect the production of exosome production, the concentration of harvested exosomes with ~150nm diameter is about 1.08 x 109/ml.
2. qRT-PCR
In the qRT-PCR experiment, we selected hsa-miR-23a-3p as the endogenous reference gene. We selected nluc as our target gene to test if the L7Ae may help to enlarge the volume of mRNA anchored on the inner surface of exosomes. We calculated the ΔCt values using the Ct of target gene (nluc) to be divided by Ct of endogenous reference gene.
The distribution of 96-well plate in qRT-PCR is displayed as below:
After the qRT-PCR, we did not get any normal signal from the group of pcDNA3.1(+)+L7Ae, nluc targeted group. In terms of the rest two groups, we analyzed theΔCt as below:
Figure 6. The ΔCt values calculated in the groups with and without C/D Box transfected.The comparison in ΔCt values between C/D Box-nluc+L7Ae groups and no-C/D Box groups has eligible difference, which indicates that the transfection of L7Ae may have limited impact in increasing the mRNA enclosed by exosomes.The characterization of L7Ae and C/D Box will be repeated in future experiments.
Measurement-Part Improvement
In this part of experiment, we repeated the experiments conducted by Peking_R 2011 team. Characterized the part BBa_k598009, BBa_k598010, and our newly designed BBa_k3030016 and BBa_k3030017. In order to get a more significant results, we improved the protocols used by Peking_R 2011 team, these improvements were well-documented in our protocols.
TWe focused on investigating the following points in our measurement-relevant experiments:
The cytotoxicity induced by theophylline to DE3 cells.
The absorbance of our cultured DE3 in different concentrations of theophylline in our experiments.
The fluorescence intensity measured that indicates the sensitivity of riboswitch to theophylline.
1. The growth curve of DE3 in different concentrations of theophylline.
We used four gradients of TPP (0 mM, 0.1 mM, 1 mM and 10 mM) to test if the addition of TPP may induce cytotoxicity to DE3, and influence the growth of them. Each single colony of the DE3 cells transfected with circularized pSB1A3 were cultured in 100 ul LB-0.1%Amp for 12.5 hours, the OD600 values were measured in each 15 minutes. We chose 12 colonies in this experiment, the cultured time was compared with the average calculated for the 12 colonies’ OD600. The growth curves of DE3 cells is displayed below:
The growth curve indicates that the OD600 measured may be influenced by the concentration of TPP, and the DE3 incubated with 1 mM TPP grows best and the 10 mM TPP may significantly inhibit the growth of DE3 cells. But the difference in OD600 was not that remarkable when the incubating time is limited in 25000 seconds. Thus, we chose to culture the DE3 cells for 6 hours in the following experiments.
2. Measuring the fluorescence intensity of DE3 cells transformed with four parts with standards and blank.
We repeated the Peking_R 2011 experiment and adapted their arrangement of TPP gradients (0 mM without arabinose added, 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM with 1 mM arabinose added). In addition, The absorbance measured was used in normalize the fluorescence intensity measured to minimize the interference caused by bacteria concentration, while the high number of colonies grew in each well may lead a wrong over-high fluorescence result. The absorbance is measured after the GFP fluorescence.
All the procedures were conducted under dark condition in a 96-well plate. The distribution of the colonies cultured is displayed as below:
(The part number of XJTLU-Natural, XJTLU-Optimized, PKU-Natural, and PKU-Engineered are BBa_k3030016, BBa_k3030017, BBa_598009, and BBa_k598010 respectively)
The GFP fluorescence values were measured, the excitation and emission light was 485 nm and 535 nm respectively. To avoid the leakage of fluorescence interfering the reading of other wells, the time interval of measurement was extended to 20 ms. The exact GFP fluorescent intensity of each well is listed below:
Figure 7. The dose responsive curves that manifesting GFP intensity measured at 535 nm divided by OD600 comparing to the TPP concentration (0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM), the concentration of arabinose in each well was controlled to be 1 mM. It can be shown that the GFP fluorescence intensity increases along with the concentration of TPP as the riboswitch was opened by the TPP.
Figure 8. The GFP/OD600 measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (*: p<0.05, **:p<0.01, ***:p<0.001).
Figure 9. The working curves that indicates the sensitivity of the riboswitch designed to TPP. The activation ratio is fluorescence intensity under given TPP concentrations compared to that of without TPP. The TPP gradients were set as 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM, the concentration of arabinose in each well was controlled to be 1 mM.
Figure 10. The activation ratio measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (*: p<0.05, **:p<0.01, ***:p<0.001).
The results shows that the riboswitch designed in part BBa_k3030017 has a 10-time expression of GFP (normalized by OD600) comparing to that of BBa_k598009, our improved part. In addition, the designed BBa_k3030017 has a 11-fold activation ratio of the absence to presence of 10mM theophylline.
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