Editing Cre VLP Optimization

== LoxP Reporter Assay using Cre plasmid<br>  ==

=== 1. CMV-Brainbow-2.1-R<br>  ===

2013-10-28<br> 

four days after transfection<br> 

{| width="795" cellspacing="1" cellpadding="1" border="1"
|-
| No<br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 7<br> 
| 8<br> 
| <br>
|-
| Clone ID<br> 
| 1522<br> 
| 1522<br> 
| colspan="2" | 1522+256<br> 
| colspan="2" | 1522+298<br> 
| colspan="2" | 1522+1400<br>
|-
| Name<br> 
| CMV-Brainbow-2.1-R<br> 
| add '''Cre VLP''' (from ~ three 10-cm plates transfected with 1400pCMV5-Gag-NLS-Cre-WPRE-oriP)<br> 
| colspan="2" | CMV-Brainbow-2.1-R+pAAV-Cre <br> 
| colspan="2" | CMV-Brainbow-2.1-R+pTriEx-HTNC (Cre)<br> 
| colspan="2" | CMV-Brainbow-2.1-R+pCMV5-Gag-NLS-Cre-WPRE-oriP<br>
|-
| color<br> 
| green<br> 
| change green to red<br> 
| colspan="2" | change green to red<br> 
| colspan="2" | change green to red<br> 
| colspan="2" | change green to red<br>
|}

<br> 

-[[Image:Cre assay1 10-28-2013.JPG]] 

=== 2. pLVX-loxP-sfGFP-loxP-TagRFP<br>  ===

four days after transfection<br> 

{| width="535" cellspacing="1" cellpadding="1" border="1"
|-
| #<br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br>
|-
| Clone ID<br> 
| colspan="2" | 1053<br> 
| colspan="2" | 1053+256<br> 
| colspan="2" | 1053+298<br>
|-
| Name<br> 
| colspan="2" | pLVX-loxP-sfGFP-loxP-TagRFP<br> 
| colspan="2" | pLVX-loxP-sfGFP-loxP-TagRFP+pAAV-Cre<br> 
| colspan="2" | pLVX-loxP-sfGFP-loxP-TagRFP+pTriEx-HTNC (Cre)<br>
|-
| color<br> 
| colspan="2" | green<br> 
| colspan="2" | change green to red<br> 
| colspan="2" | change green to red<br>
|}

<br> 

[[Image:Cre assay2 10-28-2013.JPG]] 

== HTN-Cre Activity Assay using CMV-Brainbow-2.1-R<br>  ==

HTNCre Lot:11152013<br> 

{| width="669" height="147" cellspacing="1" cellpadding="1" border="1"
|-
| <br>
| 1<br>
| 2<br>
| 3<br>
| 4<br>
| 5<br>
| 6<br>
|-
| cre concentration<br> 
| &nbsp;1uM<br> 
| 2.5<br> 
| 5<br> 
| 7.5<br> 
| 10<br> 
| 15<br>
|-
| Day 1<br> 
| <br> 
| <br>
| <br>
| 5% cell dead
| 20% cell dead<br> 
| 30% cell dead<br>
|-
| Day 2<br> 
| <br> 
| <br> 
| <br> 
| 10% cell dead<br> 
| 30% cell dead<br> 
| 60% cell dead<br>
|-
| Day 6<br> 
| <br> 
| <br> 
| <br> 
| 20% cell dead<br> 
| 50% cell dead<br> 
| 90% cell dead<br>
|-
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

Day 1 after adding CRE<br>

[[Image:11152013cre_assay_1day_2013-10-21.JPG]]<br> 

Day 2 after adding CRE<br>

[[Image:11152013cre_assay_2days_2013-10-22.JPG]]<br>

Day6 after adding CRE<br>

[[Image:11152013cre_assay_6days_2013-10-26_.JPG]]<br>

<br>

Control using&nbsp; HTN-sfGFP Lot:11082013<br>

{| width="551" height="147" cellspacing="1" cellpadding="1" border="1"
|-
| <br>
| 1<br>
| 2<br>
| 3<br>
| 4<br>
| 5<br>
| 6<br>
|-
| HTN-sfGFP concentration<br> 
| &nbsp;1uM<br> 
| 2.5<br> 
| 5<br> 
| 7.5<br> 
| 10<br> 
| 15<br>
|-
| Day 1<br> 
| <br> 
| <br> 
| 10% cell dead<br> 
| 20% cell dead<br>
| 30% cell dead
| 40% cell dead<br>
|-
| Day 2<br> 
| <br> 
| <br> 
| 20% cell dead<br> 
| 30% cell dead<br> 
| 40% cell dead<br> 
| 60% cell dead<br>
|-
| Day 3<br> 
| <br> 
| <br> 
| 30% cell dead<br> 
| 50% cell dead<br> 
| 70% cell dead<br> 
| 90% cell dead<br>
|-
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

Day1 after adding HTN-sfGFP<br> 

[[Image:SfGFP11082013_1day_2013-10-20.JPG]]<br>

Day2 after adding HTN-sfGFP<br>

[[Image:SfGFP11082013_2days_2013-10-21.JPG]]<br>

Day3 after adding HTN-sfGFP<br>

[[Image:SfGFP11082013_3days_2013-10-22.JPG]]<br>

== VLP Secrection to Medium<br>  ==

2013-11-12<br> 

GFP VLP 

1888 pCMV5-Gag-TagGFP-WPRE-oriP (HIV Gag)<br> 

3 days after transfection<br> 

[[Image:1888transfection 11-8-2013.JPG]]<br> 

<br> 

1889 pCMV5-MMLV-Gag-TagGFP-WPRE-oriP (MMLV Gag)<br> 

[[Image:1889-3transfection 11-12-2013.JPG]][[Image:1889-4transfection 11-12-2013.JPG]]<br> 

<br> 

{| width="504" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| <br> 
| colspan="3" | &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; GFP VLP supernatrant 
| <br>
|-
| <br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| <br>
|-
| <br> 
| DMEM medium<br> 
| 1888pCMV5-Gag-TagGFP-WPRE-oriP<br> 
| 1889-3pCMV5-MMLV-Gag-TagGFP-WPRE-oriP<br> 
| 1889-4pCMV5-MMLV-Gag-TagGFP-WPRE-oriP<br> 
| <br>
|-
| <br> 
| <br> 
| 7days after transfection<br> 
| 4 days after transfection<br> 
| 4 days after transfection<br> 
| <br>
|}

<br> 

[[Image:GFP VLP supernatant 11-12-2013.JPG]]<br> 

<br> 

VLP should be harvest after 7 days after transfection: see [[Virus-like particle Production|Protocol]] 

<br> 

== Transduction of GFP VLP<br>  ==

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| Cre VLP 
| GFP VLP
|-
| Day1 
| <br> 
| <br>
|-
| Day3 
| <br> 
| <br>
|}

<br> 

<br> 

== Concentrate VLP by Ultracentrifuge<br>  ==

{| width="293" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| Condition Medium<br> 
| colspan="2" | &nbsp;1 hour spin, 25000 rpm<br> 
| colspan="2" | &nbsp;2h spin<br>
|-
| <br> 
| -<br> 
| VLP pellet<br> 
| Supernatant<br> 
| VLP pellet<br> 
| Supernatant<br>
|-
| Day 1 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| Day 3 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

<br> 

== VLP Titering  ==

[[Category:VLP]]