ER-Hoxb8 system: FAQ
How stable is beta-estradiol?
It turns out that beta-estradiol (E2758) is extremely stable. I preprare solutions at 10 mM concentration in 100% ethanol and store this at -20 or -80-degrees. I add it to a final 0.5 uM (20,000x dilution) to a bottle of media and use this for up to 6-weeks.
I go through media very quickly, so I like to prepare 4 bottles at one time. I try to plan ahead and thaw all components overnight in the fridge. I have moved to purchasing the single-shot aliquots of FBS from GIBCO to avoid the hassle of thawing and re-aliquoting these and worrying about whether I have introduced any contamination. So my approach:
1. Get 4 bottles of RPMI1640 with L-glutamine.
2. Get 4 x 50 ml aliquots of FBS and put them in the fridge to thaw.
3. Get a bottle of Pen/Strep and put that in the fridge to thaw. I will keep this in the fridge for a month without free/thaw.
4. Get a bottle of L-Glutamine and put that in the fridge to thaw. This needs to be dissolved of course at 37-degrees and then I store this in the fridge for a month without free/thaw.
5. Get a tube of SCF-conditioned media (40 ml) and put this in the fridge to thaw.
The next morning prepare media:
500 ml RPMI 1640 w/ L-glutamine
50 ml FBS
10 ml SCF conditioned media
5 ml Pen/Strep
5 ml L-glutamine (yes, I add this twice, probably unecessary)
30 ul of beta-estradiol (10 mM stock in 100% ethanol)
I add the components right to the bottle and I do NOT re-filter the media at this point. I am simply careful to keep the stocks sterile.
I use this media for up to 6-weeks. I do NOT pre-heat the media before using it unless I am doing some metabolism or proliferation assay that same day. These cells do not care about cold media and I feel that this helps avoid contamination from the water bath and avoid the deterioration of the media components by leaving it at 37-degrees.
How stable is the SCF conditioned media?
This is a good question. I collected SCF conditioned media from the CHO cells and sterile filtered it and then stored it in the fridge for up to one month. I then prepared media from "fresh" conditioned media and media that had been stored in the fridge for 1-week, 2-weeks, 3-weeks, and 4-weeks. All media seemed to support the growth of the cells equivalently. My sense is that the SCF is quite stable.
What is the difference between your ER-Hox system and the ERT2-Hoxa9 system?
These systems are very similar.
Our system uses the Hoxb8 or Hoxa9 proteins fused to the estrogen-receptor hormone binding domain of the human ER-alpha. This is the G400V variant, which maintains its response to beta-estradiol but makes it MUCH less sensitive.
The ERT2-Hoxa9 system uses a tamoxifen-responsive variant of the human ER-alpha.
My sense is that these two systems provide very similar (if not complete phenocopies) systems.
Beta-estradiol has the advantage of being more stable and less toxic than tamoxifen.
Is it possible to make ER-Hox lines from human progenitors?
This is a common question and we would LOVE to have a parallel system of conditionally-immortalized human myeloid progenitors!
The short answer is "no".
To date, I have not been able to conditonally-immortalize progenitors.
I have tried a variety of starting material:
Human umbilical cord CD34+ cells
Human bone marrow CD34+ cells
Human peripherally-mobilized CD34+ cells
I have tried a few different Hox proteins, both murine and human (they are very similar at the amino acid level): HOXA9, HOXB8, NUP98-HOXA9, ER-HOXA9, ER-HOXB8.
I wonder if the problem may be one of:
- Poor gene expression from the MSCV promoter?
- Gene silencing following retroviral intregration into the genome?
- An evolutionary difference between murine and human cells in terms of their ability for transformation?
Of particular interest, it does seem that this should be possible. Dr. James Mulloy did this using the oncoprotein MLL/AF9 and I have reproduced his findings (
Mulloy JC, Wunderlich M, Zheng Y, Wei J. Transforming human blood stem and progenitor cells: a new way forward in leukemia modeling. cc. 2008 Nov 1;7(21):3314–9.) Of course, this is an oncoprotein and is not conditionally-inactivated. I have tried adding the ER hormone binding domain to both the N-terminus of MLL/AF9 as well as the C-terminus of MLL/AF9 and have not succeeded in generating a conditional version of the protein. The protein is very large, and I wonder if this is simply a technical hurdle to overcome.
I welcome ideas and collaborations! It would be a great tool for the field to have a human system of conditional myeloid immortalization.