Hi science,
As part of my PhD I need to perform transduction of RAG-/- mouse bone marrow with lentivirus for subsequent transplantation and with the goal of obtaining a transgenic population of dendritic cells. I trawled through some literature and came across a rather conflicting set of protocols, specifically for the requirement of the lin- purification prior to transduction as well as the cytokine mix. In the latter, Stem cell factor (SCF) and Flt3-ligand are routinely used, but additionally you get mentions of Il-3, Il-6, Il-11 and TPO, with no specific indication as to why they get added..
So my questions are the following:
Is lin- selection a requirement and can it be replaced by FACS sorting?
What, in your experience, is the optimal cytokine mix for my purpose and do any of them promote the myeloid/DC lineage specifically?
Many thanks!
Vitaly.
Hi Vitalijs,
We do a lot of LV transductions in my lab. I would start transducing lin- BM by MACS uisng FLT3L, TPO, SCF (FTS, as we call it). You can add other cytokines such as angiopoietins, bFGF (as in the STIF or STIFA cocktails), but for your purpose in which maintenace of HSCs is not key, I would start with FTS.
We culture the cells (optimal density: 0.5 to 1*106 cells per ml) in StemSpan + Pen/strep and stimulate with cytokines:
FLT3L 50 ng/ml final concentration
TPO 10 ng/ml final concentration
SCF 100 ng/ml final concentration
Good luck!
Hi Frank,
Thanks for your reply! I have a couple follow up question:
Do the lin- BM cells need to be cultured for a certain period or is it best to transduce and transplant as fast as possible (i.e. after overnight culture with virus).
What is the role of TPO in this mix? (And is the role of IL-6 in some protocols is to induce TPO production?)
Many thanks,
Vitaly.
Hi Vitalijs,
For gamma retroviral transductions the first day or so is of no use, as the cells need to divide before the virus can integrate. With LV you might already get transduction in more or less resting cells, keeping in mind the half-life of your virus. You may want to add a new aliquot of virus the next day. It depends of course on your question (high transduction, or single integration per cell), target cells and the virus.
Regarding the TPO, although the name thrombopoietin suggest it should only work on platelets, the TPO-R is actively expressed on HSCs. TPO therefore also is a good growth factor for HSCs. IL6 also works on HSC (especially on not so pure populations, containing MPPs as well) and helps in driving proliferation, but at the cost of differentiation. I would not add it, unless it does not matter that you lose all your stem cells.
Frank J T Staal
Hi Dr. Staal,
I isolate mouse bone marrow from tibias and femurs, do red blood cell removal, and use the media containing murine IL3, IL6 and SCF to culture bone marrow cells for 1 day before infection.
And I transfect 293T cells using pMSCV system:
(1) 0.4 mL Opti+4µL Lipo2000
(2) 0.4 mL Opri+2.5 µg pMSCV-vector+2µg pHIT/EA6x3+2µg pHIT60
Combine (1) and (2) following 30min incubation
Add the mixture in the 293T, 24h later change 10%FBS DMEM without antibiotics
48h post transfection concentrate virus using PEG reagent
Use concentrated virus to infect bone marrow cells twice, each time for 24 h.
But the GFP+ bone marrow cells are only 2.8%
Could you please kindly tell me how to improve infection efficiency? Appreciate it!
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