No tumor formation in xenograft model - (May/28/2007 )
i need some advice on what to do.
this is a prelim investigation into tumor growth...
i have 10 nod/scid mice, with a 0.72mg estrogen pellet located between shoulder blades... afer 2 days the mice were xenografted with 5 X 10 ^6 MCF-7 cells (containing an inducible plasmid). The cells were grown in normal conditions, trypsinised, spun down, washed, resuspended in sterile PBS, counted on the hemocytometer, re-adjusted so there was the right number of cells in 200uL of PBS. This was injected sub cutaneously into the left flack of the mice.
problem is... 4 weeks later, there is no tumour growth.
i first did this with rag knockout mice... they developed an inflamed uterus, but no tumours. with the nod/scids, no tumours, and they too are developing the inflamed unterus. a few of them (4/10) have very questionable tumor growth; it can't be seen, and if you feel around the site of injection, there may be something, perhaps the size of a grain of salt.
what is going on? or better still, can someone see something that i'm doing wrong?
After you injected the tumor suspension subcutaneously, did you see a little lump?
If not, you might have injected them IP...which means the tumor is inside the mouse.
Hope this may help.
it was a perfect bump just under the skin...
it just doesn't make sense to me.
Have you innoculated the parent MCF-7 cells (without any transfection) and seen tumor growth? I guess you have cells transfected with vector containing the gene of interest and also with control vector. Probably the vector transfection changed the phenotype of the cells.
You can also try mixing the cells with metrogel.
Have you checked the viability of the cells after injection? The needle could disrupt the cells if you are unlucky... Just push some cells through the syringe into a petridish and stain with tryphan blue.
Also, sometimes the mice do not form tumors... Have you tried it in nude mice? They should get some but even then there are no guarantees...
What g needle did you use?
I normally use 21or 23g to inject the cancer cells subcutaneously.
ok, 1. currently trying plain old ordinary cells... though other labs are having the same problem. it seems to be the cell line. i'm going to try a completely different cell line instead.
2. in the process of buying matrigel. apparantly this is pretty tricky to work with. any advices?
3. checked the cell viability after being syringed. all were ok.
4. i use a 21 g needle.
oh, i love the little mices, but i wish they'd get caner. sounds so wrong.
thanks for all the input.
Concerning matrigel, be sure to thaw it on ice at 4 degrees celcius overnight and don´t allow it to get warm (like room temp.) that should make it easier to handle...
I share your distress by the way... 1 in 3 mice of mine did not get tumours using one cell line but 1 of 6 when using another... Some cells apparently just don´t like it.
dumb ?, but we're all grasping at straws here....were your cells happy, young, and in early log with a good high density in the flask?
we use these models too (different cells, obviously). I can tell you that if you don't get it in nod/scids, you shouldn't see it in nudes; they have much more immune function
the year before I started in this lab, supposedly all the sudden the tumors just stopped taking well. the take-rate went down, like, 50% or more...they tried everything to get it to work. finally, they switched mouse vendors. it seems that there may have been a mutation somewhere, or something like that...perhaps the colony at the old vendor developed enough immune system to prevent the engraftment. sounds paranoid, I know, but I guess immediately after switching vendors the take rate when right back up. so, where do you get your mice?
yaa probably all of the above tips are really worthwhile. i am planning on some animal experiments next week and i decided too give a dose of 600cGy to the nude mice and do the cell line injection 24h later. this would drastically immune compromise the mice further and improve the tumor uptake.
good luck to you all and me too!