Retroviral transduction of cells - troubleshooting help please! - (Jul/27/2006 )
I am working with a Dental Pulp Stem Cell (DPSC) line - similar to mesenchymal stem cells
I am trying to stably transfect the cells with a plasmid that has been encased by a Retrovirus. For virus packaging I use 293T cells and two other plasmids that encode for the viral proteins. I also use Lipofectamine 2000.
After harvesting of the supernatant, I add 2mL virus and 1 mL serum to 6 cm plates with the DPSCs at about 40-50% confluence. I also add polybrene.
After removing the virus, allowing cells to recover, splitting and selecting the cells transfected with my control neomycin plasmid demonstrate ok survival. The cells transfected with my other plasmids all die.
Modifications/tests I have tried:
1. Double sequential virus application with standard medium application in between
2. Reduction of antibiotic concentration for selection (control did fine at either concentration)
3. No antibiotic selection - all cells grow, do western blot, probe for HA tags
Results: No tags detected, cells were not transformed
4. Agarose gel of plasmids
Bands appear to be of appropriate weight for all
5. Transient transfection of plasmids into 293T cells
All were successful and bands were present when probed on western blot
Conclusions:
It appears that the the plasmids other than the control are not being incorporated into the genome of the DPSCs.
Questions:
What else can I try to optimize this virus transfection? What might be causing my problem? Any other tips/tricks?
Thank you!
Expression of neomycin resistance gene could b low, or ur viral titres could b low.
Do u have cPPT sequence in ur vector. This helps to increase expression of the transgenes.
Do u have cPPT sequence in ur vector. This helps to increase expression of the transgenes.
Hi Malik
I'm not sure about the cPPT sequence actually. My boss handed me the vectors about a month ago before leaving town.... he didn't have a map of the construct available. He gets back on Monday so I'll ask him about it.
I thought that the resistance gene might be an issue - so after I transformed and split the cells I made one plate with each vector that did not get any neomycin. Then, I checked these plates for the transgene HA tags using a western blot.
I didn't see any tags on the blot - so, I don't think the cells were transformed regardless of the resistance gene.
For a more general question -
How does the content of a vector affect its ability to be packaged into a virus?
Do u have a fluorescent marker to assess infection of ur virus.
I guess u r having low titres or ur titres r low for the amount of cells u r trying to infect.
Anyway, check if u have cPPT. If not, then try to add it, it certainly does boost expression.
I guess u r having low titres or ur titres r low for the amount of cells u r trying to infect.
Anyway, check if u have cPPT. If not, then try to add it, it certainly does boost expression.
I do not have a fluorescent marker unfortunately, success can be determined only if the cells live. Then.... if they actually survive and grow I can probe for the HA tag.
Even if I have low titres, how common is it that no cells would survive? Shouldn't there at least be a few that get transfected?
Ok, u will have a few cells infected but the numbers could b too low and add low expression to it. U will kill all cells with the antibiotic selection.
Ok.
I think I'm going to go with the idea that my virus titres were too low. I'm going to try repackaging the plasmid in a 293T cell line that already expresses the viral proteins - while supplementing with additional viral protein plasmids, Lipo 2000, and sodium butyrate after 24 hours.
That should help me increase my titres, then I'll try the transfections again. Wish me luck!
Good Luck !!!
If u could get some virus from other source to check if they work on these cells and also to check antibioic resistance might b good.