iGEM 2018 — Team NJU-China

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# Protocol

## Primary Cortical Neuron Culture--Dissociated via Trypsin Poly-D-Lysine (PDL) Substrate:

### 1Xstock:

Add 333.35mL of sterile ddH2O to 50mg of PDL
Mix and filter through a 0.22 micronfilter
Aliquot and store at-20°C
Use immediately or within in 1-2 weeks
Only freeze / thaw once

### Coat plates:

Thaw PDL in 37°C water bath
Filter through a 0.22micr on filter
Incubate coated plates at 37°C for at least 2 hours (Best if left overnight)
1-2hours before beginning the dissection, remove the Poly-D-Lysine via vacuum and wash twice with sterile ddH2O
Allow plates to dry completely

### Dissection / Pre-preparation:

Warm the following in a 37°C H2O bath:
Hanks Balanced Salt Solution(HBSS)
0.25%Trypsin
DNase
cFBS
Preparation media (for washes, trituration, etc...)
Plating media
Add 7mL of warm HBSS to three 60mm culture dishes and 10mL of HBSS to a 15mL conical tube.
Asphyxiate an E17 timed pregnant rat by CO2 and sacrifice by cervical dislocation.
Spray lower abdomen with 70%EtOH and cut through skin and muscle with a pair of scissors exposing the uterus, intestines and embryos.
Cut embryos from amniotic sac and decapitate. Place all heads in the first dish of HBSS.
Remove brain from skull, separate the lobes( place in dish two),remove the meninges( place in dish three) and dissect the cortices (place in 15 mL conical tube containing 10 mL of HBSS).

### Cell Preparation:

Gently spin the conical tube containing the cortica ltissue at 1000rpm for 1minute.
In the tissue culture hood, remove the HBSS from the tissue and replace it with 5mL of 0.25% trypsin.
Add 75μLof0.1%DNAse(2000Units/mg) to the trypsin/tissue.
Digest tissue in a 37°C water bath for 20minutes.
After digestion, split the tissue into two new 15mL conical tubes ( you can do this by eye). One conical tube will be prepped in 3mM NB-A and the other in 25mM NB-A
If you only want one glucose concentration, no splitis necessary...
Gently spin the tubes at 1000 rpm for 1 minute.
Remove the Trypins/DNase mix from the tissue and wash with 5mL of cFBS(to inactivate the enzyme)
Leave cFBS on the cells for about 2 minutes
Gently spin the tubes at 1000 rpm for 1 minute.
Remove the cFBS and add 5mL of the corresponding NBA(either 3 or 25mM glucose)
Gently triturate the tissue with a10mL plastic pipette (about15times).
Triturate again with a flamed tip, glass Pasteur pipette(about10times).
Run the cell solution through a 70 micro strainer and into a 50mL conical tube.
Gently spin at 1500rpm for 5 minutes to pellet cells.
Remove NB-A media and re-suspend in 20mL of NB-A plating media
Dilute cells for counting by adding 20μL of cells to 180μL of plating media (10xdilution)
Add 10μL of the above cell dilute onto each side of ahemocytometer
Count cells in the 1mm center square (=25squares, each containing16smaller squares) on each side. The average = # of cells x 105/mL
To calculate the total # of cells/mL = (the average count per square) x (the dilution factor) x (105).
 Ex=78cellspersquarex10x105=780x105/mL=7.8x106 cells/mL. Therefore, the total number of cells obtained from the prep = 7.8x106 cellsx20mL=156x106 cells
Dilute the 20 mL stock to make a final concentration of 0.5 x 106 cells/mL (50% density)
Plate cells using the following volumes:
o For96wellplates o 4chamberslides o 24wellplates  o 6wellplates o T25flasks

### Restorative Feedings:

Feedcellsevery3-4days.
Always do a 50% change of media, never a 100% change.
Account for evaporation when keeping cells for long periods.

## transfection of primary neurons

For transfection, 1 μl of lipofectamine 2000 or lipofectamine LTX and 1 μg of DNA were added to 25 μl of Neurobasal medium individually and incubated at room temperature for 5 minutes. The two parts were then mixed and incubated at room temperature for another 25 minutes before adding onto each coverslip containing cultured hippocampal neurons. Culture medium was replaced with conditioned medium (fresh medium mixed with 4-day-old culture medium at 1:1) the next morning.

## Plasmid transfection and extraction of exosomes

HEK 293 cells were cultured in 25 cm2 flasks(Biofil) at 37℃, 5% CO2.When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids pmR-mCherry, pmR-mCherry with 5’utr, pmR-mCherry with 3’utr using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS.

## Exosome total RNA extraction by trizol

1. Add 0.8ml trizol to exsomes, shake and let it stand at room temperature for 5mins.
2. Add 1/5 volume of chloroform, shake vigorously and let it stand at room temperature for 3mins.
3. Centrifuge at 16000g for 10min( precool the centrifugal machine to 4℃)
4. Transfer the supernate to a new EP tube, add isopropyl alcohol in equal volume, let it sit for more than 60mins at -20℃。
5. Centrifuge at 16000g for 10mins, discard the supernate, wash the sediment with 75% DEPC ethyl alcohol.( repeat blowing and beating the sediment)
6. Centrifuge at 16000g for 10mins,discard the supernate, upside down the EP tubes until the ethyl alcohol is volatized.
7. Add DPEC water to solve the sediment( The volume of DEPC water depends on the sediment, usually 20ul-50ul)
8. Measure RNA concentration and adjust it to about 1000ng/ul, stored at -80℃。

## RNA reverse transcription and Qpcr

### RNA reverse transcription

i)Taqman reaction system(10ul system, adjust RNA concentration to 1000ng/ul in advance):
AMV: 0.5ul
AMV buffer:2ul
dNTP:1ul
DEPC water: 4.5ul
Taqman: 1ul
Template RNA: 1ul
Target RNA and reference RNA reaction system need to be constructed individually.
ii) Reverse transcription
Edit the protocol as follows:
16℃ 15mins
42℃ 60mins
85℃ 5mins
4℃ store

### Qpcr Taqman reaction system(20ul per hole)

10X buffer: 2ul
dNTPs: 0.4ul
MgCl2: 1.2ul
rTaq:0.3ul
ddH2O:14.77ul
Taqman: 0.33ul
cDNA: 1ul

## fluorescence in situ hybridization

### Cell Fixation

(1) Cells were 80–90% confluent at the time of fixation.
(2) Remove growth media and dissemble chambers.
(3) Submerge the slides in a staining dish containing 1X PBS.
(4) Remove 1X PBS and add 10% Neutral Buffered Formalin (NBF). Incubate at ROOM TEMPERATURE (RT) for 30 min.
(5) Remove NBF and gently rinse slides with 1X PBS. Repeat twice.

### Dehydrate and Store Cells

(1) Remove final 1X PBS wash and replace with 50 mL 50% EtOH. Incubate at RT for 5 min.
(2) Remove 50% EtOH and replace with 50mL 70% EtOH. Incubate at RT for 5 min.
(3) Remove 70% EtOH and replace with 50 mL 100% EtOH. Incubate at RT for 5 min.
(4) Remove 100% EtOH and replace with fresh 100% EtOH. Incubate at RT for 10 min.

### Rehydrate Cells

(1) Submerge slides in 70% EtOH. Incubate at RT for 2 min.
(2) Remove 70% EtOH and replace with 50% EtOH. Incubate at RT for 2 min.
(3) Remove 50% EtOH and replace with 1X PBS. Incubate at RT for 10 min.

### Create a Hydrophobic Barrier

(1) Draw 2–4 times around each well/circle on the chambered slides using the ImmedgeTM hydrophobic barrier pen. Let the barrier dry completely ~1 min.
(2) Rinse slides briefly with 1X PBS in a staining dish.

### Apple RNAscope Protease III(320850, ACDbio)

(1) One at a time, remove each slide from the 1X PBS and tap/flick to remove excess liquid. Place the slides on the HybEZTM Slide Rack and place rack in the Humidity Control Tray.
(2) Add 2–4 drops diluted Protease III to completely cover each well/circle.
(3) Close the Humidity Control Tray and incubate for 10 MINN at RT.
(4) One at a time, take each slide from the HybEZTM Slide Rack and tap/flick to remove excess liquid. Submerge slides in 1X PBS.
(5) Wash the slides by agitating them in the 1X PBS. Repeat with fresh 1X PBS.

# Calendar

1. [1 Apr](#0)
2. [8 Apr](#0)
3. [15 Apr](#0)
4. [22 Apr](#0)
5. [29 Apr](#0)
6. [5 May](#0)
7. [27 May](#0)
8. [10 Jun](#0)
9. [24 Jun](#0)
10. [1 Jul](#0)
11. [7 Jul](#0)
12. [15 Jul](#0)
13. [22 Jul](#0)
14. [29 Jul](#0)
15. [5 Aug](#0)
16. [12 Aug](#0)
17. [19 Aug](#0)
18. [26 Aug](#0)
19. [2 Sep](#0)
20. [9 Sep](#0)
21. [16 Sep](#0)
22. [23 Sep](#0)

* [Prev](#0)
* [Next](#0)

1. ## Week 1

   *04/01-04/07*

   Team member recruitment

   First meet up
2. ## Week 2

   *04/08-04/14*

   Team brainstorming

   Establishing team project orientation
3. ## Week 3

   *04/15-04/21*

   Background survey

   Discussion with tutors
4. ## Week4

   *04/22-04/28*

   Project design modification and optimization
5. ## Week5

   *04/29-05/05*

   School capstone presentation
6. ## Week 6-8

   *05/06-05/26*

   Learn laboratory safety regulations and lab skills

   Visited the local brain hospital and further determined the significance of the project
7. ## Week 9&10

   *05/27-06/09*

   Plasmids design and construction based on pmR-mCherry ( two experimental groups with the TBEV-5’-UTR or the b-Actin-3’-UTR separately and the other one with the two elements combined)
8. ## Week 11&12

   *06/10-06/23*

   Plasmid transformation, amplification and extraction
9. ## Week 13

   *06/24-06/30*

   Primary Cortical Neuron Culture （3days）
10. ## Week 14

    *07/01-07/07*

    Transfect pmR-mCherry, pmR-mCherry with 5’utr, pmR-mCherry with 3’utr into Primary Cortical Neuron. 2 days later test cell viability and trace mCherry’s red florescence of each group. Fix the neurons with formalin.
11. ## Week 15

    *07/08-07/14*

    Location of exogenous RNA by fluorescent in situ hybridization.
12. ## Week 16

    *07/15-07/21*

    HEK293 cell line recovery
13. ## Week 17

    *07/22-07/28*

    HEK293 cell proliferation for mass-production of exosomes

    Design and order of AAV virus
14. ## Week 18

    *07/29-08/04*

    Transfect pmR-mCherry, pmR-mCherry with 5’utr, pmR-mCherry with 3’utr into HEK293.

    Collect exsomes from the cultural media of HEK293.
15. ## Week 19

    *08/05-08/11*

    Exosomes nanosight.
16. ## Week 20

    *08/12-08/18*

    Determination of total protein in exsomes.

    Extract RNA from exsomes, reverse trascripte RNA.

    Test the relative level of mRNA in total RNA by Q-PCR.
17. ## Week 21

    *08/19-08/25*

    AAV stereotactic injection of mouse brain
18. ## Week 22

    *08/26-09/01*

    Attend CCIC (Conference of China iGEMer Community) and communicate with other Chinese teams
19. ## Week 23

    *09/02-09/08*

    Brain tissues were fixed and photographed with fluorescence.
20. ## Week 24

    *09/09-09/15*

    Model built.
21. ## Week 25

    *09/16-09/22*

    HP：organize a public benefit activity, using VR glasses to let the general public feel the suffering of psychiatric patients，arrange donation to contribute to the rare mental illness.
22. ## Week 26&27

    *09/23-10/06*

    Results comprehensive analysis and model application.

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# Team:NJU-China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:NJU-China/Results

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## Cell viability

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.

![](https://static.igem.org/mediawiki/2018/7/72/T--NJU-China--2018--R1.png)

## 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](https://2018.igem.org/Team%3ANJU-China/Model))

![](https://static.igem.org/mediawiki/2018/5/56/T--NJU-China--2018--R2.png)

## 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.

![](https://static.igem.org/mediawiki/2018/2/21/T--NJU-China--2018--R3.png)

## Exosomes

C

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.

## NTA

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.

![](https://static.igem.org/mediawiki/2018/6/6e/T--NJU-China--2018--R5.png)

## qPCR

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.

![](https://static.igem.org/mediawiki/2018/a/aa/T--NJU-China--2018--R6.png)

## 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.

![](https://static.igem.org/mediawiki/2018/6/6d/T--NJU-China--2018--R7.jpeg)

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