Lentiviral Transduction Troubleshooting - Transducing Raji + Daudi with pLentiCRISPR v2

[Michael J Cargill]

I am having trouble getting lentiviral infection of two Burkitt Lymphoma cell lines -- Raji and Daudi -- using pLentiCRISPR v2 (and a couple v1) constructs containing sgRNA's. Here's an overview of my protocol:

* Plasmids are mini-prepped with multiple preps pooled per plasmid.

* cRPMI = RPMI 1640 + 10% FBS + L-Glut + HEPES + Pen/Strep

Day 0: Seed a 6-well plate with ~7.5E5 293T cells in 2 mL cRPMI

Day 1: Transfect ~60-80% confluent 293T cells in 6 well plate with XTREMEGene HP transfection reagent with the following complex after 20 min room-temp incubation: 1200 ng pLentiCRISPR, 900 ng pdeltaVpr, 300 ng pVSV-G in 180 uL OptiMEM + 6 uL HP reagent 

Day 2: Replace media with cRPMI (since the viral supernatant will be used for suspension cell transductions)

Day 4: Harvest lentiviral supernatant and passed through 0.45 um filter, 1E6 Raji/Daudi cells are re-suspended in harvested viral supernatant and spinoculated in 8 ug/mL polybrene, 2220 rpm, at 30 degrees for 45 minutes

Day 5: Replace media with cRPMI

Day 7: Raji/Daudi are selected with 2 ug/mL Puromycin for three days whereupon assays begin.

Unfortunately, I am unable to get any of my Raji or Daudi cells to survive puro selection. 

I have cloned in an emGFP gene into the pLentiCRISPRv2 backbone and have been able to generate about 40% GFP from just the XTREMEGene transfection (after 48 hrs) of 293T. However, I am unable to get a GFP signal from my Raji/Daudi cells 72 hrs post-transduction.

Does anyone know of easy pitfalls I may be falling into? I am trying to keep my protocol as is since I am trying to replicate an experiment that followed this protocol. The only internal steps are doing the lentiviral production in cRPMI and the selection concentration was determined empirically. 

I would really appreciate words of lentiviral wisdom!!

[Phil Abbosh]

Hi Michael
qiagen Miniprep manual says preps can have 1200 U endotoxin per ug of DNA.  In contrast, the qiagen midiprep kit that I use says preps can have 1 U endotoxin per ug of DNA.  I know some people transfect miniprepped plasmid but it feels wrong to me.

I transfect at 4:4:2 lentiCRISPR:packaging:VSVG

I collect infectious media 48h after transfection. waiting longer makes the media look super-spent.

I usually infect with 500 uL of clarified infectious media with 2 mL of regular media for the corresponding cell line in polybrene 8ug/mL, then change media 24h later, then passage 24h after that at a low density in puro.

I dont use spinfection, just gravity.

I usually get 90+% GFP+ cells and almost always do a GFP lentivirus alongside to reassure me things are working.

I will also admit to having a mycoplasma problem recently that really constipated the process. now that I am clean again everything works. I didnt think it could happen to me.  strongly recommend checking if you havent.

phil

[Carter]

Promega, zymo, omega and probably some other companies sell mini prep kits with endotoxin removal steps. The plasmid preps are suitable for transfection. The zymo kit works well plus it's cheaper and faster than the QIAGEN kit.

[Mark]

I was able to derive Raji Cas9 cells pretty easily without spinfection.

Have you verified if you're actually packaging virus? Your transfection being green only tells you that your promoter works --- maybe your entire backbone (or your packaging vectors) is recombined that's why you can't package it? Maybe try to dump some of your supernatant onto cells that you know is easily transducible (293T).

[Michael J Cargill]

Thanks, all! When I miniprepped my plasmids, I sequenced the key lentiviral elements on the plasmid and did a restriction digest and all looked good. Still, I am in the process of midiprepping the lentiviral plasmids with PureLink instead. 

I recently measured GFP by flow of some Raji cells that were transduced with emGFP (72 hrs post-transduction) and only got ~1% GFP by flow. However, the pLentiCRISPR-emGFP was packaged in 293T cells which showed >90% GFP (by flow) on the day of lentiviral harvest. So my three possibilities for a low transduction efficiency are:

1.) Mycoplasma

2.) Old polybrene (the stock is about 16 months old that has been stored at 4 degrees)

3.) Transduction seeding density 

I feel fairly confident that lentivirus is being produced, so I believe the actual transduction step is where things go awry. I will try to play/check with the above factors, but are there any other red flags that jump out to people?

Thanks again SO much for all of your insight - I feel so close to getting substantial transduction efficiency!

-Michael

[Michael J Cargill]

PS. I am also in the process of transducing 293T cells as well. Should know more later this week about that.

[Mark]

Might be #3. I usually have my B cells at 2-3X10^5 cells/mL the day before so they're right in log phase when you're ready to transduce (4-6x10^5cells/mL).

I don't think it's #2. We've been using the same diluted stock stored in 4C for 2 years and still works for us.

Good luck!

[Phil Abbosh]

I actually wonder how much the polybrene adds.  i have forgotten to add before and it seemed like it made no difference. maybe thats why it "works" with old polybrene.  think about it...in nature, the virus probably isnt dependent on polybrene.

phil

[Mark]

It's cell line dependent. I've worked with other cell lines that really need it to get a decent % of transduction (from <5% to 70%). 

Also, adding too much polybrene is toxic to some cells.

[Michael J Cargill]

OK so...

I tried transducing 293T with the lentiviral supernatant (the producing cells from which had >90% GFP at time of harvest) and measured their GFP content 72 hrs post-transduction, but only got about 5% GFP + cells. My hunch is that the viral titer isn't very high and that I have too many target cells relative to the viral titer, thus I will try varying the seeding density to see if that helps. I'm hoping for around 20% transduction efficiency at the time of puromycin selection to get enough cells for my Cell-Titer Glo assay. 

I also have been seeding my suspension cells the day of and not the day before (as I have noticed many protocols do with suspension cell lines), so I will try that as well. Also, the mycoplasma testing results came back negative.

Hopefully the seeding density variation along with the use of endotoxin-free midiprepped plasmids will help increase the transduction efficiency!

Thanks for any and all advice :)

Michael

[Moli Joshi]

Hi Michael,

Did you figure out the issue with transduction?

-Moli

[Michael J Cargill]

Not really. Had to use lenti-x to concentrate virus, but still got pretty poor transduction efficiency with Cas9 containing constructs.

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Michael J. Cargill

Warren Lab

PhD Candidate - Molecular Medicine and Mechanisms of Disease (M3D)

 
Phone: (360) 431 - 3211
Alt Email: mcar...@fhcrc.org 

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I have titrated the puro concentration for Daudi before, it was around 0.3ug/ml. I think your puro concentration might be too high!

Afina

On Friday, August 5, 2016 at 8:44:41 PM UTC-4, Michael J Cargill wrote: