Team:Heidelberg/Notebook/Mouse Infection

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=Mouse Infection=
 
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=October=
 
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==15/10/2010==
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;"
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* organize 600 Million HEK293T cells
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|- border="0"
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* prepare 2 liter of media
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! colspan="7" style="background:#78b41e;" | [https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October<font color="white">October</font>]
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==17/10/2010==
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|- style="background:#78b41e; color:white"
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* plate 100 15cm dishes with each 6*10^6 cells
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|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
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==18/10/2010==
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|- style="background:#f2f2f2; color:#78b41e"
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* due to contamination 70 new plates are seeded in the afternoon
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|colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#03.2F10.2F2010 3]'''
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==19/10/2010==
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|- style="background:#f2f2f2; color:#78b41e"
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* triple transfection with PEI using standard protocol for triple transfection:
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#05.2F10.2F2010 5]'''
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* set-up of the plate:
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#10.2F10.2F2010 10]'''
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** 1) off target: construct M23 with perfect binding site for miR122, AAV8 serotype (AAV8), Adenohelper virus 5 (Ad5)
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|- style="background:#f2f2f2; color:#78b41e"
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** 2) control: pBSU6Sv40Luc2 transgene in ITRs transfected together with shuffeled capsid of the 50 clones which was capable of transducing Huh7 and HepG2 cells, Ad 5
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#12.2F10.2F2010 12]'''|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#15.2F10.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#17.2F10.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#18.2F10.2F2010 18]'''
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** 3) control: pBSU6sv40Luc 2 transgene, AAV8, Ad 5
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|- style="background:#f2f2f2; color:#78b41e"
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** 4) shRNA miR expression vector for miRhaat (pBSU6H1haat), AAV8, Ad 5
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#19.2F10.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#20.2F10.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#21.2F10.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#22.2F10.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#23.2F10.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#24.2F10.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#25.2F10.2F2010 25]'''
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** 5) the same as 4)
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|- style="background:#f2f2f2; color:#78b41e"
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** 6) tuning construct: perfect binding site for shRNA miRhaat (M1), AAV8, Ad5
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#26.2F10.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#27.2F10.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#28.2F10.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#29.2F10.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#22.2F30.2F2010 30]'''||colspan="3"|
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** 7) tuning construct: imperfect binding site randomized from nucleotide 9-12 (M9), AAV8, Ad5
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|- style="background:#f2f2f2; color:#78b41e"
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==21/10/2010==
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|colspan="7"|
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* harvest 70 plates of Hek cells
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<span style="color:#ffffff">-</span>
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* centrifuge: 10 min at 1500 rpm
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|}
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* wash with 30ml 1xPBS
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* centrifuge: 10 min at 1500 rpm
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* start freeze and thaw cycles
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* pour the gradient
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* centrifuge in the ultracentrifuge for 2h at 10°C and 50K
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* soak out the purified virus out of the 40% phase
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----
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==22/10/2010==
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* double check the titre of capsids via dot plat and the packaged DNA via viral DNA extraction and running on an agarose gel using a virus with a known titre as a control
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* mice were injected the following:
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* 1) SV40-Luc2-miR122 perfect binding site in AAV8 capsid
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* 2) SV40-Luc2 in our shuffeled capsid
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* 3) SV40-Luc2 in AAV8 capsid
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* 4) SV40-Luc2-miRhaat perfect binding site in AAV8 capsid
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* 5) SV40-Luc2-miRhaat imperfect binding site (9-12) in AAV8 capsid
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* 6) Sv40-Luc2-miRhaat perfect binding site double injected with H1-shhaat both in AAV8 capsid
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* 7) Sv40-Luc2-miRhaat imperfect binding site double injected with H1-shhaat both in AAV8 capsid
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==25/10/2010==
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*plate 40 15 cm dishes with 5.5*10^6 cells per dish
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==26/10/2010==
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*transfect all 40 plates using PEI transfection buffer and the following set-up
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==27/10/2010==
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* bioluminescence measurements on mice from the first injection round
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{{:Team:Heidelberg/Pagemiddle}}
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{{:Team:Heidelberg/Bottom}}
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{{:Team:Heidelberg/Side_Bottom}}
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__NOTOC__
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= in vivo Study =
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<html>
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<map name="2" id="2">
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<area shape="rect" coords="260,25,353,91" href="#AAVcomparison" alt="" />
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<area shape="rect" coords="252,198,364,277" href="#AAVcomparison" alt="" />
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<area shape="rect" coords="85,339,180,407" href="#miR122" alt="" />
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<area shape="rect" coords="491,287,586,357" href="#hAAT" alt="" />
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<area shape="rect" coords="380,367,474,437" href="#hAAT" alt="" />
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</map>
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<img src="https://static.igem.org/mediawiki/2010/3/3d/Mouse_data_for_Wiki.png" width="618" height="464" border="0" alt="" title="" usemap="#2" /><br>
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<p>Figure 1: Scheme of luciferase
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<br><br><br><br><br>
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{{:Team:Heidelberg/Single_Bottom}}

Latest revision as of 04:00, 28 October 2010


in vivo Study


Figure 1: Scheme of luciferase




{{:Team:Heidelberg/Single_Bottom}}

Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection"