Cre VLP Optimization

LoxP Reporter Assay using Cre plasmid

1. CMV-Brainbow-2.1-R

2013-10-28

four days after transfection

No	1	2	3	4	5	7	8
Clone ID	1522	1522	1522+256		1522+298		1522+1400
Name	CMV-Brainbow-2.1-R	add Cre VLP (from ~ three 10-cm plates transfected with 1400pCMV5-Gag-NLS-Cre-WPRE-oriP)	CMV-Brainbow-2.1-R+pAAV-Cre		CMV-Brainbow-2.1-R+pTriEx-HTNC (Cre)		CMV-Brainbow-2.1-R+pCMV5-Gag-NLS-Cre-WPRE-oriP
color	green	change green to red	change green to red		change green to red		change green to red

-

2. pLVX-loxP-sfGFP-loxP-TagRFP

four days after transfection

#	1	2	3	4	5	6
Clone ID	1053		1053+256		1053+298
Name	pLVX-loxP-sfGFP-loxP-TagRFP		pLVX-loxP-sfGFP-loxP-TagRFP+pAAV-Cre		pLVX-loxP-sfGFP-loxP-TagRFP+pTriEx-HTNC (Cre)
color	green		change green to red		change green to red

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

HTNCre Lot:11152013

	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

Day 2 after adding CRE

Day6 after adding CRE

Control using  HTN-sfGFP Lot:11082013

	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

Day2 after adding HTN-sfGFP

Day3 after adding HTN-sfGFP

VLP Secrection to Medium

2013-11-12

GFP VLP

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

3 days after transfection

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

		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

VLP should be harvest after 7 days after transfection: see Protocol

Transduction of GFP VLP

	Cre VLP	GFP VLP
Day1
Day3

Concentrate VLP by Ultracentrifuge

	Condition Medium	1 hour spin, 25000 rpm		2h spin
	-	VLP pellet	Supernatant	VLP pellet	Supernatant
Day 1
Day 3