We first constructed three kinds of plasmids with mCherry report gene, the control, ones with TBEV 5’-UTR and those with the β-Actin 3’-UTR. To test the transportation efficiency in neural cells, we first need to confirm that our elements do no harm to the experimental cells. Thus, the cell viability test is followed. We monitored the cell viability with absorbance value after 48 hours of transfection. With data collected, we created a graph to show the difference, and there is no significant difference in the viabilities of neurons in the three groups. Thus, further study of the elements in test on neurons could be conducted.
mCherry protein into the dendrites
We did some experiments to see the transportation efficiency of the elements in vitro. With the help of mCherry’s fluorescence, the position of the protein could be tracked in the cells. More protein fluorescence at the further end in the dendrites could be an indicator that the transportation efficiency is higher. By comparing the images from the control and experimental groups, we can conclude that the protein could have a larger distribution along the dendrites with the transportation elements and TBEV 5’-UTR has better transportation efficiency. Models are also constructed to prove the conclusion quantitatively.(Model)
mCherry mRNA in the dendrites
However, a main drawback in the fluorescent experiment is that there is also protein transportation system inside cells, which means the presence of the protein could be a result of this system rather than mRNA transportation. We then carried out the mRNA tracing experiment with in situ hybridization. Several figures are gathered and if we merge the ones from mRNA tracing with those in protein fluorescence, the presence of the protein and that of the mRNA are found to correlate with each other. Our conclusions are therefore re-assured. Besides, we could also consider the protein fluorescence mostly as an indicator of mRNA transportation and local translation.
With respect to our past work around the exosomes, we first expect to pack the mRNA into the exosomes for in vivo tests. Some experiments are also done in this stage.
We performed the nanoparticle tracking analysis (NTA) to have a more precise determination of the quantity and size of the secreted exosomes. With certain analysis tools, the mean diameter of the extracellular vesicles （EVs） is calculated to be around 142.9nm, which means that most of the secreted EVs are exosomes we need.
After collecting the exosomes, the first thing we would like to know is whether the target mRNAs we need were packaged into exosomes and whether the concentration of target mRNA could meet the demand. The qPCR experiment helps to solve these problems. The cycle number from qPCR data is negatively correlated to the original concentration of the mRNA substrates. With charts shown, the TBEV 5’-UTR groups run more cycles, meaning that the concentration of the TBEV 5’-UTR group is rather low. Therefore, other packaging materials should be introduced for further use of this element.
TBEV test in vivo
To discover the physiological and pathological mechanisms and promote the further practice of our element, we carried out the experiment in vivo. Due to the advantages of virus packaging, we customized the AAV virus containing the gene sequence with TBEV 5’-UTR from a biological company. After virus injection, the mouse brains were analyzed. From the photograph of the brain slices, we could primarily conclude that the AAV virus is capable of locating the mRNA to the brain tissue effectively and this package does not influence the performance of our elements in mRNA transportation.