8/13/2019 Danijela January 31, 2014
6/6
Previously in the monkey storm animals we identified several
mCherry and Luciferase positive cells and somewhat less BFP and
Luciferase positive
cells (6 BFP/Luciferase cells were found in total from two
animals). However we found many more BFP cells that were very
bright in blue channel,
were completely negative in green channel but were negative for
Luciferase. We were confortable saying that the Luciferase staining
was not the
issue since we would find mCherry/Luciferase positive cells in
the same tissue sections. Nonetheless these cells were so nice
looking with perfect
and quite strong distribution of BFP signal right around the
nucleus. So in order to f igure out why we are not seeing
Luciferase signal in BFP positive
cells Deirdre infected some 293T cells with these 3 viruses:
a) Lich Generation 2, containing mCherry and Luciferase
reporters,
b) Lich Generation 3, containing mCherry, paGFP and Luciferase
reporters and
c) Lich Generation 3, containing BFP, paGFP and Luciferase
reporters.
She stained all of these cell types with Luciferase that were
labeled with Zenon 647 and Draq5 nuclear stain. She also had all of
the cell types stained
only with Draq5 nuclear stain to have a control for non-specific
Luciferase staining. We imaged all of these on the microscope and
found out the
following:
-When cells were infected with Lich Generation 2, containing
mCherry and Luciferase reporters, we get very robust Luciferase
expression (10%T and
0.1 ms around 3000 max reading), robust mCherry expression (32%T
and 0.3 ms around 3000 max reading) and at least by half lower
readings in
TRITC channel (32%T and 0.3 ms around 1000 max reading).
Confirming that these cells were Luciferase/mCherry high and TRITC
low. To be sure
that mCherry signal is real and not bleed through from the
strong reading in cy5 channel I also looked at the slides that got
Gen2
(mCherry/Luciferase) infected cells but were only stained with
DRAQ5 (so no Luciferase staining) and once again saw comparable
levels of mCherry
vs TRITC. No photo activation is observed in these cells, as
expected since there is no paGFP in gen2 construct. So all good
here.
-When cells were infected with Lich Generation 3, containing
mCherry, paGFP and Luciferase reporters, we see very similar
pattern and levels ofmCherry/Luciferase/Tritc. These cells had low
level of GFP increase following the photo-activation which is much
less than we previously observed in
tissue culture cells but in tissue sections we see variable
degree of photo activation ranging anywhere from 10-50 percent.
=When cells were infected with Lich Generation 3, containing
BFP, paGFP and Luciferase reporters, we find many BFP positive
cells. BFP levels were
through the roof (10-32%T and 0.1-0.3ms conditions resulted in
maximum intensity of close to 4000). These cells were all
completely negative in
FITC channel (32%T and 0.3 ms resulted in maximum reading of
100!). However these cells were also completely negative for
Luciferase, much like
we are seeing in our recent tissue samples. I also found some
cells that had average BFP expression and very strong emission in
FITC channel (again
something we find in our tissue, and consider false-positive
cells).
-Puzzled by this finding we decided to see if we can find the
very bright blue cells among the cells that were infected withLich
Generation 2,
containing only mCherry and Luciferase reporters and I found
several that were extremely bright in Blue but negative in green
and cy5 channels.
-All of these findings got us questioning if the virus made was
even expressing Luciferase at the first place. However infectivity
of this virus was doneusing Luciferase readouts and I as I
mentioned earlier we gound six BFP/Luciferase positive cells in two
of the Monkey Storm animals.
-The more time that we spent looking at these bright blue cells
and comparing them to the mCherry/Luciferase cells from the Gen2
infections the
more we began to feel that these cells do not look quite right
While cells containing gen2 and gen3 cherry viruses had very nice
long extensions in
which mCherry and Luciferase signals were robust, the bright
blue cells did not have these long extensions and the blue staining
was appeared to be
very concentrated in the nuclear region Since this batch of gen3
blue cells did not look very healthy to allow us to reliably test
the luciferase
expression in BFP positive cells, we decided to infect more 293T
cells with this vector hoping that once we see healthy looking
cells with long
extensions that we will see BFP and Luciferase being
expressed.
-In the mean time we are continuing to look and phenotype
mCherry/Luciferase and BFP/Luciferase positive cells in our tissue
sections for several of
the priority animals, so far analyzing the tissue from animal
M301 did not lead to identification of any more mCherry/Luciferase
or BFP/Luciferasecells from what was reported in the previous blog
by both Deirdre and me.
