GenY HSA:iRFP670 Maxi Prep

Diagnostic Digest Maxi prep DNA was Made

3kb
2kb
1.5kb
1kb

1,800bp

my Mike and purified with

Cesium Chloride (1.95g/l
conc.)
Digestion with Ecor5 and
Sal1 is suppose to drop
down the
IRES_HSA:iRFP670 insert
band of 1,800bp
We used this DNA to make
VSVg virus to transduce
293T cells and monitor
luciferase and iRFP670
expression
Gen2 iRFP670 construct
was used to make control
VSVg virus for comparison

293T Transduced Cells with Gen2

iRFP670 VSVg Virus

Luciferase:F
ITC

iRFP670
(direct)

Conclusion: Cells transduced with Gen2 iRFP670 virus express

both Luciferase and iRFP670 proteins robustly.

Merge

293T Transduced Cells with GenY

HSA:iRFP670 VSVg Virus

Luciferase:F
iRFP670
ITC
(direct)
Conclusion: Cells transduced with GenY HSA:iRFP670 virus
express Luciferase robustly but no iRFP670.

Merge

293T Transduced Cells with GenY

HSA:iRFP670 VSVg Virus

Luciferase:F
ITC

iRFP670
(direct)

Conclusion: Cells transduced with GenY HSA:iRFP670 virus

express Luciferase robustly but no iRFP670. Same conclusion as
on the prior slide.

Merge

293T Transduced Cells with GenY

HSA:iRFP670 VSVg Virus

HSA:FITC
Conclusion: Cells transduced with GenY HSA:iRFP670 virus also
express HSA robustly. So in overall conclusion Luciferase and HSA are

Gen2_iRFP670 vs.
GenY_HSA:iRFP670 Sequence
Comparison
Gen2_iRFP670 Sequence

ACCGGT: Age1 Site

GTCGAC: Sal1 Site
iRFP670 sequence (start and stop sites
present)

GenY_HSA:iRFP670 Sequence

ACCGGT: Age1 Sites

GTCGAC: Sal1 Site
GCCGCCACC: Kozak Sequence
HSA Sequence (start site present, stop site
deleted)
iRFP670 sequence (start site deleted, stop
site present)