Lich Generation 2 (mCherry/Luciferase)

mCherry

TRITC

Luciferase:Cy5

mCherry Luciferase:Cy5

Lich Generation 2 (mCherry/Luciferase)

Photo activation Profile of the two cells on previous slide. No difference before and after photo activation as expected in Generation 2 vectors.

Lich Generation 3 (mCherry/paGFP/Luciferase)

mCherry

TRITC

Luciferase:Cy5

Before PA After PA

Lich Generation 3 (BFP/paGFP/Luciferase)

BFP

FITC

BFP LUCIFERASE

BFP

FITC

BFP LUCIFERASE

Lich Generation 2 (mCherry/Luciferase)

Very bright blue junk in gen2 infected cells (10 %T and 0.1 ms resulted in max intensity of over 4000), signal was only observed in blue channel.

mCherry

Luciferase:Cy5

DAPI channel

mCherry Luciferase:Cy5

mCherry Luciferase:Cy5 DAPI channel

DAPI channel

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 figure 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 of mCherry/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 with Lich 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 done using 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/Luciferase cells from what was reported in the previous blog by both Deirdre and me.