Difference between revisions of "Part:BBa K2619101:Experience"
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<h4>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:</h4>
 
<h4>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:</h4>
 
<p></p >
 
<p></p >
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[[File:L7Ae-1.png|thumb|left|400px|]]
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[[File:L7Ae-1.png|thumb|center|400px|]]
 
<p></p >
 
<p></p >
 
<h4>We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:</h4>
 
<h4>We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:</h4>
 
[[Media:File:L7Ae-1.pdf]]
 
[[Media:File:L7Ae-1.pdf]]
 
<p></p >
 
<p></p >
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[[File:Glutamate2.png|thumb|center|600px|'''Figure 2:''' The comparison in glutamate concentration between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells, student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).]]
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[[Media:File:L7Ae-2.pdf]]
 
<p></p >
 
<p></p >
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<h4>The results of glutamate assay reveals that the expression of EAAT2 on cell membrane can maintain the concentration of glutamate in a relatively stable level, and significantly reduce the concentration of glutamate in the cell medium. Higher the glutamate concentration is, the more significant the EAAT2 may transport the glutamate in cells. In addition, the expression of EAAT2 will not over-transport the glutamate and make the glutamate concentration lower than the origin level.</h4>
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[[Media:File:L7Ae-3.pdf]]
 
<p></p >
 
<p></p >
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<h2>2.WST-1 Cytotoxicity Test</h2>
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<h4>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.</h4>
 
<p></p >
 
<p></p >
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<h4>To detect the cytotoxicity induced by the over-high glutamate concentration, we used WST-1 as an alternative of MTT in our test. The dehydrogenase in mitochondria can reduce the WST-1 to soluble and chromatic formazan, and a high absorbance measured indicates a low cytotoxicity induced by glutamate. The absorbance of these samples are measured at 450 nm and 690 nm as a reference wavelength.
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<h4>For the particles with different diameters, the distribution of their concentration in each set of samples are listed as below:</h4>
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The distribution of each sample are displayed as below:</h4>
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[[File:NTA-1.png|thumb|center|600px|]]
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[[File:NTA-2.png|thumb|center|600px|]]
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[[File:NTA-3.png|thumb|center|600px|]'''Figure 1:''' The distribution of particles with different diameters measured by NTA in a 50 ul ultracentrifuged exosome solution 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.]
 
<p></p >
 
<p></p >
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[[File:WST-1.png|thumb|left|400px|]][[File:WST-2.png|thumb|right|400px|]]
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<h4>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.</h4>
 
<p></p >
 
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[[File:WST-3.png|thumb|center|600px|'''Figure 3:''' The absorbance (A450) measured after with reference absorbance (A690) and blank value subtracted, and its correlation to treated duration.]]
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<h2>2. qRT-PCR</h2>
 
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[[File:WST-4.png|thumb|center|600px|'''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), student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).]]
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<h4>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.</h4>
 
<p></p >
 
<p></p >
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<h4>The results of WST-1 indicated that the expression of EAAT2 on cell membrane can 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.</h4>
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<h4>The distribution of 96-well plate in qRT-PCR is displayed as below:</h4>
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[[File:L7Ae-4.png|thumb|center|400px|]]
 
<p></p >
 
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<h1>Further Application</h1>
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<h4>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:</h4>
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<h4>Our primary goal is to save astrocytes from the high glutamate level at the early stage of neurodegenerative disease and strengthen the defence against the neurotoxicity. In details, we design this therapy to enhance the glutamate absorbing function by overexpression of EAAT2 on the astrocyte’s membrane. We use the mRNA-based vector for expression and protect the mRNA by exosome through the delivery process. We will further study the production, characterization, transportation, preservation and other related content of exosome therapy, and subsequently, carry out social activities related to exosome treatment to enhance our design.</h4>
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[[File:L7Ae-5.png|thumb|center|400px|'''Figure 2:''' The ΔCt values calculated in the groups with and without C/D Box transfected.]]
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<p></p >
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<h4>This results indicated that the transfection of L7Ae may slightly increase the RNA anchored on the exosomes, but the difference inΔCt between the C/D Box transfected and untransfected were not significant. The characterization of L7Ae and C/D Box will be repeated in future experiments.</h4>
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===User Reviews===
 
===User Reviews===
 
<!-- DON'T DELETE --><partinfo>BBa_K2619101 StartReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K2619101 StartReviews</partinfo>

Revision as of 16:11, 21 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2619101

Background

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: 1. The production of exosome in exosome donor (HEK293T cells) 2. The RNA anchored in the exosome

Experiment data

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:

L7Ae-1.png

We sent 50 ul of exosome isolated from each set of cells, the results were sent back and listed below:

Media:File:L7Ae-1.pdf

Media:File:L7Ae-2.pdf

Media:File:L7Ae-3.pdf

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:

NTA-1.png
NTA-2.png

[[File:NTA-3.png|thumb|center|600px|]Figure 1: The distribution of particles with different diameters measured by NTA in a 50 ul ultracentrifuged exosome solution 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:

L7Ae-4.png

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 2: The ΔCt values calculated in the groups with and without C/D Box transfected.

This results indicated that the transfection of L7Ae may slightly increase the RNA anchored on the exosomes, but the difference inΔCt between the C/D Box transfected and untransfected were not significant. The characterization of L7Ae and C/D Box will be repeated in future experiments.

User Reviews

UNIQe160cca6669ac67d-partinfo-00000000-QINU UNIQe160cca6669ac67d-partinfo-00000001-QINU