do you use polybrene and plate centrifuge for the transduction?

both of them are important.

Junjie Shao - I add 8ug/ul of polybrene to the virus prior to adding. I have not tried centrifuging the plate. I use 6 well plates at the moment containing 1x10^5 of seeded HEK293 cells.

thanks for your suggestions!

Hi Scot,

I suggest you centrifuge your cells as Shao suggested. I add 5ug/ul of polybrene directly to the virus media and then mix very well. Wash the cells once with PBS and add the mixture (virus media with polybrene) directly onto the cells (caution: HEK cells may come off from the plate thus preferably add it on the wall of the wells). I sealed the plate with parafilm and centrifuged for 90 min at 2500 rpm.

Alternatively, if you have the lentivirus plasmid and VSVG together with gag/pol plasmids you may easily transfect your HEK cells using PEI (polyethyleneimine). I am happy to help if you require a protocol for this.

Any details on the vector and construct?

It's a self-inactivating lentiviral plasmid for co-expression of my gene of interest and EGFP ( Empty backbone ). http://www.addgene.org/21373/

To start with I want to optimise the transduction process using an empty backbone, then purchase the same backbone but with my gene of interest later down the line.

Khamal Ampah - Thanks for your suggestions, have you found transfecting HEK cells using a PEI (polyethyleneimine) method to work better or just as good?

Scot, using PEI, I do achieve greater than 90% efficiency especially with lentivirus transfection in HEK cells.

What is the promoter used in lentiviral vector, it might be a clue of the problem (if the gene of interest is not toxic).

https://www.researchgate.net/post/Protocol_for_stable_lentiviral_transduction_of_U937_HL60_or_K562_cell_lines

Article A Comparative Analysis of Constitutive Promoters Located in ...

The CMV promoter in your vector is known to be silenced in some cases (ex. in in hematopoietic cell lines). Try switching your promoter to a EF1, ubiquitin or CAG. Can You use antibiotic resistance including vector? Once I had plenty resistant cells but none of them were green- (CMV issue)

Now I get it is about HEK cells that should transduce pretty instantly, and with CMV promoter express a lot of gene of interest. IMHO there is sth wrong with lentivirus preparation.

Also, as You asked, excessive integration (multiplicity of infective unitis) might lead to siliencing

https://www.vectalys.com/blog/is-gene-expression-permanent-in-target-cells-after-several-divisions-230

Are the cells producing the virus green (should be very)?

Hi Scott,

From my experience, in most cases of low infection efficacy the reason is the transfection. As Adam asked: did the transfected cells produced strong green florescence? and also how did the culture looked before the infection (in term of viability and confluency).

I did experience one case of too much virus in HEK cells but it was very obvious that such high concentration was simply toxic.

Good luck

Hi Adam,

I should have mentioned, we requested the supplier to replace the CMV promotor with an EF1 promoter. I am not too sure on an antibiotics marker to use.

In our lab, I used created some lentivirus with a vector that had a UBC promoter.

previous lab members have used this for past projects and I performed this to use as a positive control to see if my lentiviral packaging had issues. ( This was separate to my main vector I posted previously. These cells had turned green using this vector. I use EVOS to image the cells and FACS to measure the GFP.

Did the cells generating the lentivirus with the gene of interest (fused to GFP?) turn green? How do You check plasmid quality?

I did not assess whether the lentivirus producing HEK cells turned green, unfortunately. I assessed my transfer plasmid using restriction digest and right bands appeared so the plasmid appeared correct.

Which kind of HEK cells You use for those two purposes?

Isn't transfection enough?

I would suggest checking the florescence level of your virus producing cells (if the plasmid is marked) as standard, It's really helpful. I did noticed that in good transfection the virus producing cells look very bad, which allows me to get a general feeling for the virus titer. Lastly I think you will see cytotoxicity due to high viral load before it will effect the transduction. What were the transduction percentages (for each concentration)?