Dear Sreeja,

I would highly recommend two alternatives: a chemical one and a "physical" one.

• The first one would be a direct substitute to Polybrene: LentiBlast. It neutralizes electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Moreover, due to a favorable “membrane permeable effect” limiting the transmembrane potential changes it is non-toxic and totally compatible with cell viability.

For your convinience you can find in attached files some results with LentiBlast, you can refer to page 3 for viability assays compared to polybrene.

• The second one would be the Magnetofection technology *** with ViroMag and ViroMag RL. These two magnetic particles formulations bound to viral particles and allow a dramatic increase in infection and transduction.

The main advantages of Magnetofection is that, once the viral particles are complexed to magnetic nanoparticles, you can concentrate them with a magnet, remove supernatant, and resuspend them in smaller volume to concentrate up to 1000 times your initial viral suspension.

I'm pretty sure these reagents will give you reliable efficiency, you can contact me directly at [email protected] if you need more informations or if you want to try either LentiBlast or Magnetofection.

Good luck for your experiments,

Cedric

*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).

http://www.ozbiosciences.com/7-viral-applications-transduction-transfectionreagents

Thanks Cedric.

What is your transduction efficiency? You may be at the limit using retroviral transduction... this is something cell specific, using 1 round of transduction (you may choose to repeat) it isn't unusual to only get 40-60% efficiency.

There are a few things to consider:

Retroviral vectors only transduce dividing cells. Seed MEFs that are dividing well (do not grow to confluency as they will slow down with contact inhibition) and seed these at a suitable density for transduction (I seed my target cells to be ~10% confluent at time of adding vector supernatant). Grow them to confluency before analysing efficiency. It may increase further with another 1-2 passages.

Have you checked you have reasonable titre? If it is low, check the %GFP of your transfected 293T cells 1 day after transfection - you should have 70-90% strongly GFP+. I use PEI and find it very reliable (and cheap). Calcium phosphate is also popular because it is cheap, but it is very fussy with pH and how you make the precipitate. Definitely important to monitor transfection efficiency with CaPO4.

Concentrating your vector by ultracentrifugation (or reagents e.g. clontech Retro-X if you don't have an ultrafuge)  will only allow you to add higher amounts of vector in a limited volume. I routinely concentrate by ultrafuge so that I can save aliquots at -80degC with known titres, but if you just want to transfer supernatant and make fresh each time, the titre should be high enough that concentration is not required.

Spinoculation can help. Add the vector to cells in a multiwell dish and spin the plate for 45 minutes at 1500g. This improves my transduction rates (at low MOI) ~5 fold. Of course if you are the maximum efficiency already, adding more vector and spinoculating won't make any difference.

As Cedric says, polybrene helps with VSV-G pseudotyped vectors.

Good luck

Thanks Nathan for your valuable suggestion.I was using MEF cells pf passage number 25 with 70-80% confluency.Does the passage number affects the efficiency?I will go for 10-20% confluency next time as you suggested.

Hi Sreeja,

I suspect that passage number can affect efficiency (up or down) but I very much doubt this is a significant factor for you.

Certainly 70-80% confluency is going against you and 10-20% will help to ensure a high % of cells are happily dividing. Although you did not confirm if you have checked you have adequate titre or at least high 293T transfection efficiency at the stage of producing the vector. Also, you didn't say what your current transduction efficiency is.

All the best,

Nathan

I have 30-40% transfection efficiency in HEK 293 cells.The maximum transduction efficiency which I got was 14%.One more question I want to ask is that does the duration of viral supernatant  incubation affects the efficiency?I am incubating MEF cells with 1 ml sup.+10ug/ml DEAE dextran for 4 hrs,then replenishing the media with regular DMEM(in one well of 6 well pllate)