Hello everyone,
I am currently working with a virus that I purchased from Dharmacon that has an inducible shRNA for my gene of interest under the control of a TRE promoter. The construct also has inducible GFP expression. I transduced the benign breast cell lines MCF-10A and MCF-12A with this virus and am growing them under a 1ug/ml concentration of puromycin. However, when I try to induce shRNA expression through the addition of doxycycline, there is almost no induction of GFP expression or shRNA expression. But the cells are thriving under puromycin, which indicates that there is genomic integration of my construct.
What could be the reason for this? I am using the Smart Vector Inducible shRNA product from Dharmacon.
PS: I have already tried different MOIs of transduction and also did a dose analysis with different dox concentations.
Thanks!
Hello Akhila.
There may be following possibilities for the concurrent undetectable GFP expression and no induction of shRNA.
MCF10A cells are modestly dipoloidy, but MCF12A cells are polyploidy, having 65-71 chromosomes. It would be a good idea to test only in the MCF10A cells first since they are more likely having a single copy of your target gene.
1) The number of viral gene copy incorporated may be possibly low, thus surviving under the puromycin concentration. However, low gene copy number inserted can't give detectable GFP and shRNA. You need to make maximum number of gene copies be inserted until you see bright GFP expression. A single or a few gene copies would not give visible GFP expression under fluorescence microscope.
2) The culture systems for both cell lines require a bit special conditions that may have forced the cells to be activated all the time regardless of Dox addition to the point of maximum threshold of gene activation, not allowing further extra gene activation. For test this possibilities, the prospective transfectant cells should be maintained in minimal medium without special supplements that can constitutively activate the cells for 18h. And then test your Dox for indcution of GFP.
3) There are many unproven, but experienced discussions in similar Q&A of RG. That is when additional or prolonged Dox treatments are used, general gene activation is halted or downregulated in RG.
Regards.
Hello Dr. Lee,
Thank you for your suggestions, they make a lot of sense. I will make sure to try them out!
What do you mean almost no induction? Do you see some GFP in all cells, some cells?? Check your virus plasmid by transient transfection into HEK cells. Does your plasmid carries also the RTA or you need co-infection with a second virus? If the second is the case obviously you have not incorporated the RTA plasmid.
Hello Dr. Papamatheakis,
What I mean by almost no induction is that only 10-15% of my cells express GFP post treatment with dox, and very faintly. The construct has the RTA element too, so a second plasmid is not required. Thanks!
Ιn this case you can increase the puromycin. Alternatively you can select individual clones or sort for fluorescence. MCF cells have this problem especially when the virus titer is low.
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