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| | <h1>AAV</h1> | | <h1>AAV</h1> |
| - | <p>We choose adeno-associated virus as platform to construct plasmids, which could just insert into 19 chromosome. Then we designed the following experiments to prove it safe and practicable.</p> | + | <p>We choose <i><b> adeno-associated virus </b></i> as <i><b> platform </b></i> to construct plasmids, which could just insert into 19 chromosome. Then we designed the following experiments to prove it <i><b> safe and practicable.</p> |
| | <p>1. Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )</br> | | <p>1. Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )</br> |
| | 2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.</br> | | 2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.</br> |
| - | 3. Transfect the mCherry-AAV into 293T cell line through calcium phosphate-based method. During this step the plasmids, pAAV-RC and pHelper, which express the shell of AAV are needed. (this kind of method that divide the virus into more than one expressing plasmids makes AAV safe)</br> | + | 3. Transfect the <i><b> mCherry-AAV </b></i> into 293T cell line through calcium phosphate-based method. During this step the plasmids, pAAV-RC and pHelper, which express the shell of AAV are needed. (this kind of method that divide the virus into more than one expressing plasmids makes AAV safe)</br> |
| | 4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.</br> | | 4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.</br> |
| | 5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry. | | 5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry. |
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| | <div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/2014_thu_aav_fig3.png" width="350" height="350"></br><span style="font-size:10px" >Figure3. 293T cell after merge</span></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/2014_thu_aav_fig3.png" width="350" height="350"></br><span style="font-size:10px" >Figure3. 293T cell after merge</span></div> |
| | <p>We can see from the figure that recombinant mcherry-AAV vectors express in the 293T cell successfully, which prove that the AAV system is practicable.</br> | | <p>We can see from the figure that recombinant mcherry-AAV vectors express in the 293T cell successfully, which prove that the AAV system is practicable.</br> |
| - | However, the system is not exactly perfect. Although when 293T cells are producing virus we can detect 70%-80% positive rate, after virus infect the 293T cells, less than 1% positive rate can be observed. We have suspected that the pAAV-RC and pHelper plasmids are wrong, so we do an enzyme-cleavage experiment to test them, the results are as follows:</p> | + | However, the system is not exactly perfect. Although when 293T cells are producing virus we can detect <i><b> 70%-80% positive rate </b></i>, after virus infect the 293T cells, <i><b> less than 1% </b></i> positive rate can be observed. We have suspected that the pAAV-RC and pHelper plasmids are wrong, so we do an enzyme-cleavage experiment to test them, the results are as follows:</p> |
| | <div align="center"><img src="https://static.igem.org/mediawiki/2014/f/fe/2014_thu_aav_fig4.jpg" width="350" height="280"></br><span style="font-size:10px" >Figure4. RC1 mono-enzyme cleave the pAAV-RC</span></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2014/f/fe/2014_thu_aav_fig4.jpg" width="350" height="280"></br><span style="font-size:10px" >Figure4. RC1 mono-enzyme cleave the pAAV-RC</span></div> |
| | <p>The result match the DNA sequence we expect, so the plasmids are right.</br> | | <p>The result match the DNA sequence we expect, so the plasmids are right.</br> |
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| | Another condition can’t be ignored that we use 293T as the virus-producing platform, but in fact, there are various 293 cell lines, such as 293F, 293A and etc. Therefore, 293T cell lines may not be the best cell line to produce the AAV virus.</br> | | Another condition can’t be ignored that we use 293T as the virus-producing platform, but in fact, there are various 293 cell lines, such as 293F, 293A and etc. Therefore, 293T cell lines may not be the best cell line to produce the AAV virus.</br> |
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| - | Then we sort the 293T cells which have mcherry fluorescence, and after incubate the cell as many as enough, we can test the expression of mcherry on the mRNA level by qPCR.</p> | + | Then we sort the 293T cells which have mcherry fluorescence, and after incubate the cell as many as enough, we can test the expression of mcherry on the <i><b> mRNA level </b></i> by qPCR.</p> |
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AAV
We choose adeno-associated virus as platform to construct plasmids, which could just insert into 19 chromosome. Then we designed the following experiments to prove it safe and practicable.
1. Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )
2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.
3. Transfect the mCherry-AAV into 293T cell line through calcium phosphate-based method. During this step the plasmids, pAAV-RC and pHelper, which express the shell of AAV are needed. (this kind of method that divide the virus into more than one expressing plasmids makes AAV safe)
4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.
5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry.
The results are as follows:
 Figure1. 293T cell under mcherry fluorescence |  Figure2. 293T cell under whitefield |
Figure3. 293T cell after mergeWe can see from the figure that recombinant mcherry-AAV vectors express in the 293T cell successfully, which prove that the AAV system is practicable.
However, the system is not exactly perfect. Although when 293T cells are producing virus we can detect 70%-80% positive rate , after virus infect the 293T cells, less than 1% positive rate can be observed. We have suspected that the pAAV-RC and pHelper plasmids are wrong, so we do an enzyme-cleavage experiment to test them, the results are as follows:
Figure4. RC1 mono-enzyme cleave the pAAV-RCThe result match the DNA sequence we expect, so the plasmids are right.
Another condition can’t be ignored that we use 293T as the virus-producing platform, but in fact, there are various 293 cell lines, such as 293F, 293A and etc. Therefore, 293T cell lines may not be the best cell line to produce the AAV virus.
Then we sort the 293T cells which have mcherry fluorescence, and after incubate the cell as many as enough, we can test the expression of mcherry on the mRNA level by qPCR.
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