Team:Tsinghua/Project/Virus

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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 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>
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    <ul>
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  <p>1.   Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )</br>
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    <ol>1. Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )</ol>
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  2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.</br>
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    <ol>2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.</ol>
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     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>
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     <ol>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)</ol>
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  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>
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    <ol>4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.</ol>
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     5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry.  
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     <ol>5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry.  
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The results are as follows:
The results are as follows:
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</ol>
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</br>
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     </ul>
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     </p>
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Revision as of 16:25, 17 October 2014

Team Tsinghua 2014 Return to iGEM 2014

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:

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