Hello everyone, I need suggestions to circumvent some silly problems I am facing with my recent experiments involving lentivirus transduction. This particular construct (transfer vector) is about 12 kb long with LTR to LTR distance is around 10.2 kb.
Method for producing lentivirus:
I use 293T lenti X cells in 6 well format. On the day it becomes ~70% confluent, I transfect 1ug pMD2 + 2.5 ug pSPax + 3 ug transfer vector using X-fect transfection reagent (Clontech). After 48 hours of transfection I generally add 1 ml fresh media per well in order to keep the proper pH of the media. So, after about 60-70 hours post transfection, I collect the media by passing it through 0.45 um PES filter and add lenti X concentrator @1:3 ratio. Usually keep it at 4c for atleast 5 hours before spinning it down at 1500Xg for 45 minutes. Remove the sup and resuspend the pellet in appropriate volume of DMEM to get 4-8 times concentrated lentivirus. Then add it directly to the cells containing 8ug/ml polybrene. Keep it for 48 hours before changing it with fresh media.
The problem:
I had transduced equal volume of lentivirus prepared this way to HEK293T lenti X cells as well as SH-SY5Y cells (my target cells) for 48 hours. I do not know the titter of the lentivirus preparation. I got considerable amount of green signal (transfer vector has EGFP) from HEK cells (roughly about 70-80%) but very few green cells I could see in SH-SY5Y cells. Although I haven’t count the transduction efficiency in SH-SY5Y cells, but it can be estimated to be ~10% after 48 hours. Moreover, the signal intensity is also extremely dim in SH-SY5Y cells as compared to bright signal in HEK cells.
Another problem I found is, it is expected to have to “nuclear signal (puncta pattern)” upon expression of this construct in the cell, but I don’t see much nuclear signals both in HEK293TlentiX cells as well as in SH-SY5Y cells. The bright signal what I saw in HEK cells, majorly came from cytoplasm. Only ~10% of the HEK cells show desired nuclear bright puncta pattern out of total 70-80% green transduced cells. Now, I would like to mention here that, when I am transfecting this construct during lentiviral preparation (or during any other transfection-based experiments), 100% of the time I see 80% nuclear puncta signals along with very few cells showing cytoplasm-only signals. Cells are not unhealthy or dyeing post transduction.
I would sincerely appreciate if anyone can suggest me some way out or can point out some problem with my method. I will try to understand any reasonable explanation of this unexpected behavior of my lentivirus. Has anybody used different serotypes for transducing SH-SY5Y cells? Do I have less MOI thats why I dont see nuclear signal? Remember one more thing, this SH-SY5Y cells I can transduce with other regular lentiviruses having smaller size backbones. And in those instances, I usually get very high transduction efficiencies. Therefore, I prepare this known lentiviruses along with my “problem making one” as positive control for virus preparation.
Thanks in advance!!!!
1) It's very normal to observe different transduction efficiencies between cell types. There's a lot of potential reasons, but one major reason is likely that different cell types express differing levels of LDL receptor, which is the ligand that VSV-G protein uses to enter the cell.
2) In my experience with Lenti-X concentrator, I've noticed that the concentrated virus tends to disrupt lipid membranes, I often see syncytia forming when use this reagent. My guess is that this is probably due the presence of PEG or PEG-like compounds in the concentrator reagent itself. If you can, the best way to concentrate is to use an ultracentrifuge.
hey it may be easier to transduce neurons using pseudotyped vectors with other glycoproteins. Here is an example you may find in Emory University:
Article Comparative analysis of HIV-1-based lentiviral vectors beari...
Hi Subhrangshu,
Sometimes it can be very difficult to achieve reliable high delivery efficiency when using viral systems. May I ask if you have tried Self-Deliverable RNA silencing FANA Molecules to achieve high delivery efficiency?
If not, I encourage you to review the attached FANA delivery protocol without the use of any transfection reagents.
Please kindly let me know if this can help your specific experiments?
Have a good day!
Thanks for your suggestion. It does look interesting. Just that I need to see if this system can be customized with my construct. I am not aiming to knockdown any gene, that's why I need to see if this FANA-ASO method can be applicable for me.@Matthew Adler
@ David Lee thanks for the suggestion. Yes, the protocol/method I follow for the initial transfection in HEK293Tlenti X cells using X-fect reagent (Clontech), gives me close 100% transfection efficiency as evidenced by the presence of GFP positive cells. And as mentioned in my post, lentivirus prepared this way also produces huge amount of GFP+ cells upon transduction in HEK cells. But its not the case for SH-SY5Y cells transduction.
- Badges
- Science method