FLP VLP Optimization 2014 3 4

Making VLP

Co-transfect FRT reporter plasmid 2013 with VSVG and FLP constructs

293T cells, 12 well plate

FRT plasmid: 2013 pCMV-FRT-TurboRFP-ODC-2Stops-FRT-TurboGFP, 0.3 ug

FLP plasmid: 0.6 ug

pCMV-VSVG (505): 0.6 ug

2103 pCMV-RSV-Gag-Flpo, #1	2103 pCMV-RSV-Gag-Flpo, #2	2103 pCMV-RSV-Gag-Flpo, #3	2103 pCMV-RSV-Gag-Flpo, #1 maxi
1969 pCMV5-Gag-p2b-NLS-Flp-WPRE-oriP	1962 pCMV5-HTN-Flpo	1445 pCMV5-Gag-NLS-Flp-WPRE-oriP	1981 pCMV5-Tat-Flp-PDGFR
1964 pCMV5-n-NA-NLS-Flp-WPRE-oriP	1526 pCDNA3.1-NLS-FLPo	1525 pCDNA3.1-FLPe	GFP contro, no 2013

Co-transfect FRT reporter plasmid 2013 with FLP constructs (no VSVG)

293T cells, 12 well plate

FRT plasmid: 2013 pCMV-FRT-TurboRFP-ODC-2Stops-FRT-TurboGFP, 0.5 ug

FLP plasmid: 1.1 ug

2103 pCMV-RSV-Gag-Flpo, #1	2103 pCMV-RSV-Gag-Flpo, #2	2103 pCMV-RSV-Gag-Flpo, #3	2103 pCMV-RSV-Gag-Flpo, #1 maxi
1969 pCMV5-Gag-p2b-NLS-Flp-WPRE-oriP	1962 pCMV5-HTN-Flpo	1445 pCMV5-Gag-NLS-Flp-WPRE-oriP	1981 pCMV5-Tat-Flp-PDGFR
1964 pCMV5-n-NA-NLS-Flp-WPRE-oriP	1526 pCDNA3.1-NLS-FLPo	1525 pCDNA3.1-FLPe(cell death due to contamination)	2013 pCMV-FRT-TurboRFP-ODC-2Stops-FRT-TurboGFP

Activity assay

add 0.5 ml condition culture medium to 24 well plate, 293T cells transfected with 2013 pCMV-FRT-TurboRFP-ODC-2Stops-FRT-TurboGFP

VSVG-FLP

2103 pCMV-RSV-Gag-Flpo, #1	2103 pCMV-RSV-Gag-Flpo, #2	2103 pCMV-RSV-Gag-Flpo, #3	2103 pCMV-RSV-Gag-Flpo, #1 maxi
1969 pCMV5-Gag-p2b-NLS-Flp-WPRE-oriP	1962 pCMV5-HTN-Flpo	1445 pCMV5-Gag-NLS-Flp-WPRE-oriP	1981 pCMV5-Tat-Flp-PDGFR
1964 pCMV5-n-NA-NLS-Flp-WPRE-oriP	1526 pCDNA3.1-NLS-FLPo	1525 pCDNA3.1-FLPe	GFP contro, no 2013

FLP (no VSVG)

2103 pCMV-RSV-Gag-Flpo, #1	2103 pCMV-RSV-Gag-Flpo, #2	2103 pCMV-RSV-Gag-Flpo, #3	2103 pCMV-RSV-Gag-Flpo, #1 maxi
1969 pCMV5-Gag-p2b-NLS-Flp-WPRE-oriP	1962 pCMV5-HTN-Flpo	1445 pCMV5-Gag-NLS-Flp-WPRE-oriP	1981 pCMV5-Tat-Flp-PDGFR
1964 pCMV5-n-NA-NLS-Flp-WPRE-oriP	1526 pCDNA3.1-NLS-FLPo	1525 pCDNA3.1-FLPe	2013 pCMV-FRT-TurboRFP-ODC-2Stops-FRT-TurboGFP