Difference between revisions of "Part:BBa K3030003"

Revision as of 11:47, 21 October 2019

Excitatory Amino Acid Transporter 2 with C/D Box on pcDNA 3.1+

This part contains EAAT2 protein that may selectively transport glutamate, which is an universal excitatory neurotransmitter, from cleft and extracellular matrix. The high expression of EAAT2 can significantly alleviate the excitotoxicity induced by the overhigh glutamate. The part also contains C/D box which may binds to L7Ae transfected and expressed on inner side of exosome membrane, and help hold the mRNA of EAAT2 on exosomes.The vector of this part is pcDNA3.1+ that can be expressed in eukaryotes, which includes the promoter CMV.
Sequence and Features

Background EAAT2-Functional Characterization At the beginning of our project, we characterized the function of EAAT2 and its expression on pcDNA3.1(+), and simulated the inflammation-inducing environment for Neuro-2a cells to mimic the high-glutamate condition in neurodegenerative patients’ cerebrospinal fluid. In this part of test, we focused on two impacts our exosome medicine may bring to the patients: 1. Reducing the excitatory amino acids in cerebrospinal fluid. 2. Alleviate the cytotoxicity brought by over-high glutamate or the excitotoxicity induced. Experiment data 1.Glutamate Assay We tested two sets of cells: the EAAT2-transfected N2a cells and pcDNA3.1-transfected cells (negative control). For each set of N2a cells, the time interval for changing the DMEM medium to inflammation-inducing medium is three hours. There are four groups (approximately 0 hour, 3 hours, 6 hours and 9 hours) in total depending on the time to change the medium. The extracted cell medium was then applied with glutamate assay kit and the absorbance for each sample was read in plate reader. The distribution of each sample are displayed as below: PICTURE1 PICTURE2 PICTURE3 Figure 1. The glutamate concentration measured and calculated by comparing with calibration curve and plate blank, and its correlation to treated duration. PICTURE4 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).

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.

2.WST-1 Cytotoxicity Test 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. The distribution of each sample are displayed as below: PICTURE5 PICTURE6 PICTURE7 Figure 1. The absorbance (A450) measured after with reference absorbance (A690) and blank value subtracted, and its correlation to treated duration. PICTURE8 Figure 2. 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).

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.

Further Application