Paul.xu at 19:48, 27 November 2013

2013-11-27T19:48:51Z

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== LoxP Reporter Assay using Cre plasmid ==

=== 1. CMV-Brainbow-2.1-R ===

2013-10-28 

four days after transfection 

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

 

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

=== 2. pLVX-loxP-sfGFP-loxP-TagRFP ===

four days after transfection 

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

 

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

== HTN-Cre Activity Assay using CMV-Brainbow-2.1-R ==

HTNCre Lot:11152013 

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

Day 1 after adding CRE 

[[Image:11152013cre_assay_1day_2013-10-21.JPG]] 

Day 2 after adding CRE 

[[Image:11152013cre_assay_2days_2013-10-22.JPG]] 

Day6 after adding CRE 

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

 

Control using  HTN-sfGFP Lot:11082013 

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

Day1 after adding HTN-sfGFP 

[[Image:SfGFP11082013_1day_2013-10-20.JPG]] 

Day2 after adding HTN-sfGFP 

[[Image:SfGFP11082013_2days_2013-10-21.JPG]] 

Day3 after adding HTN-sfGFP 

[[Image:SfGFP11082013_3days_2013-10-22.JPG]] 

== VLP Secrection to Medium ==

2013-11-12 

GFP VLP

1888 pCMV5-Gag-TagGFP-WPRE-oriP (HIV Gag) 

3 days after transfection 

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

 

1889 pCMV5-MMLV-Gag-TagGFP-WPRE-oriP (MMLV Gag) 

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

 

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

 

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

 

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

 

== Transduction of GFP VLP ==

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

 

 

== Concentrate VLP by Ultracentrifuge ==

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

 

== VLP Titering ==

[[Category:VLP]]

Cre VLP Optimization - Revision history

Paul.xu at 19:48, 27 November 2013