I have a mouse model that expresses modified Cre recombinase, so that only upon tamoxifen treatment of the animals they express Cre, thus KO of floxed genes can be induced. I am frequently isolating primary pre-adipocytes and differentiating them in vitro, so I was thinking of isolating these cells from my Cre expressing mice and then treating them with tamoxifen in vitro. I was wondering if anybody can give me suggestions as to how to treat cells in culture with tamoxifen. Do I do this over the whole course of differentiation or only for 1 day after they are differentiated? Which concentrations and solvents are recommended?
For treatment of the animals I had to dissolve it in 5/6 oil and 1/6 EtOH, which probably is an insufficient solution for cell culture.
For cells in culture I have used 4-hydroxy tamoxifen instead of tamoxifen free base. It's more expensive than the free base, but worked very well in vitro. It is dissolved in methanol at 5mg/ml as a stock solution, which can be further diluted in PBS. I use a final working concentration in culture at 0.02mg/ml. This concentration has worked well in macrophages, T cells, and in morula aggregation assays for us. It shouldn't be necessary to expose the cells to 4-OH Tamoxifen for the entire course of differentiation, we get excellent deletion after 24h of treatment. Of course, the effectiveness of the particular Cre recombinase and the floxed gene itself are factors in the recombination efficiency. Hopefully this helps.
For cells in culture I have used 4-hydroxy tamoxifen instead of tamoxifen free base. It's more expensive than the free base, but worked very well in vitro. It is dissolved in methanol at 5mg/ml as a stock solution, which can be further diluted in PBS. I use a final working concentration in culture at 0.02mg/ml. This concentration has worked well in macrophages, T cells, and in morula aggregation assays for us. It shouldn't be necessary to expose the cells to 4-OH Tamoxifen for the entire course of differentiation, we get excellent deletion after 24h of treatment. Of course, the effectiveness of the particular Cre recombinase and the floxed gene itself are factors in the recombination efficiency. Hopefully this helps.
Do you have any reporter gene which confirms you if cre recombinase is expresed and hopefully the deletion has been done?
Because I was wondering whether you could perform in vivo deletion and then isolate
deleted cells and culture them later.
But I don not know if you need WT cells at the beggining of the culture.
Hi Karin,
I agree with Philip and David. I think you'll need to do an optimization experiment to see what dose and what duration of the treatment you'll need. And 4-OH tamoxifen is definetely a go. So if you can assess for your deletion by FACS/WB set up a time course and try different dilutions of -OH tamoxifen to find your optimal conditions as diddrent cells and tissues respond differently.
On the other issue could you please let me know your protocol for isolating, culturing and differntiating adipocytes.I'd like to look at the adipocytes from mouse mammary gland.
Thank you in advance
Marina
Hi Karin,
I also agree with Philip and would do a dose/response curve for different time points (12, 24 and 48h) and check for ko with PCR.
For the right time to treat your cells I would say that this depends on what you expect. When you expect that ko pre-adipocytes can not longer differentiate it maybe would be the best way to treat the pre-adipocytes with tamoxifen and start the differentiation afterwards. I know that the diferentiation takes ~ 7-10 days, and I would not treat the cells over the whole time with tamoxifen.
The best option is to use HTNCre protein transduction. It's super easy, recombination efficiency of over 90% , nontoxic, not time consuming and very cheap. I have plenty of stocks I could eventually share.
Hello everybody!
Firstly, thank you a lot for your responses. This sounds very helpful, i will try with 4-Hydroxy-TAM. Yes, I have a floxed gene, whose deletion would be a good reporter to test whether the cre-recombinase would be expressed upon TAM treatment, I could detect a 80% reduction upon in vivo injection of TAM. However I cannot isolate these cells, because I can only isolate precursor cells, however the Cre recombinase is expressed unter a tissue specific promotor that is only active in differentiated cells. This I would have to differentiate them first and then treat them in vitro.
Marina, our protocol is rather quick and dirty, we dissect the adipose depots, perform a collagenase digest, strain the cells through a 300µM mesh and plate them to laminin-coated cell culture plates. Then we let them grow 90% confluent in DMEM and then induce adipogenic differentiation (Dex, Insulin, T3). Let me know if I can provide you with further details!
German, I am not sure I understand, could you explain this method to me?
Thank you!
Hi, I take the chance of this old discussion to ask you all a question about 4OH-Tamoxifen stability (inactivation of Tamoxifen due to Z to E isomerization in common solvents, such as ethanol, methanol etc.). Did anybody use the THF (tetrahydrofuran stabilized with 0.025% BHT) to reconstitute 4OH Tamoxifen to prevent isomerization and add it to the culture media? I generally add 1ul of vehicle (ethanol up to now) for each 1ml of culture media. Might it be toxic ? Thank you in advance for your help!
Hi Everyone,
Before I get into detail, I would like to introduce myself. I am a Ph.D. candidate working on ER-alpha reexpression in TNBC cells. I wanted to use Tamoxifen for cell culture and then got suck with this confusion wither to use Tamoxifen or 4OH-Tamoxifen for cell cultures. Any comments/suggestion? Thanks!
4-OH tamoxifen is typically used in cell culture. 4-OH tamoxifen is the active form of the drug, tamoxifen gets converted to 4-OH tamoxifen in vivo.
Okay. Thanks. Just one more query: In term of stability, I guess tamoxifen is more stable than 4-OH tamoxifen?
Not sure about stability of 4-OH tamoxifen. The few times I've used it, I've made a fresh stock every time. Tamoxifen dissolved in corn oil is very stable at -20C, I've used frozen stocks more than a year after making them, with good results.
@ Rishabh: In cell culture you should always use the 4-OHT (the active metabolite). In vivo Tamoxifen gets activated and transformed by CYP2D6, usually expressed in the liver. 4-OHT is quite unstable in solution and should be protected from light. I wouldn't use stock solutions older than 2 months
Definitely 4-OHT. Before I knew that I tried regular Tamoxifen in cell culture. It does not work, not at all. Makes sense now :)
Hi
I use 4-OH tam to induce neural stem cells grown as neurospheres in vitro (0.5 and 1uM). I have a tdtomato reporter in my cells, so 24 hrs after induction I wash off the drug from the media and I can see RFP expression under the microscope. Do Cre expressing cells, express any kind of reporter in your system? in which case you can look for the expression of the reporter 24 hrs after induction. If it does express then you might not need to expose your cells until differentiation. Of course this depends on your cells type. In my systemn, once my gene is deleted in neural stem cells, all the daughter cells produced from them will also lack the gene. I don't know if you need the drug until differentiation, but I have read papers where they have left the drug in for about 2 weeks, however I don't know at what concentration. Also I use 4-OH tam from Sigma and I resuspend it in EtOH. You should know that my vehicle control cells died once, which was odd, because I resuspended the drug in 100% EtOH and used it. I later found out that 100% EtOH has benzene in it to keep it pure. So that might have affected my cells. The second time I resuspended tam in 95% EtOH and it worked fine. I did not want to use Methanol as I use stem cells and they are very sensitive. Of course this depends on your cell type. I usually use 1:9 (EtOH: Corn oil) for in vivo induction only.
Good luck!
@ German Camargo I want ask if you make TAT Cre in your lab ??
Also , Could you share your protocol for TAT Cre deletion ??
Which cells have you tried the treatment on ?
@Philip E Lapinski After treating T cells with 4-hydroxy tamoxifen , did you get downregulation of surface markers like CD8, TCR ??
Namrata:
I never looked at expression of surface markers. This will have to be determined empirically.
@ Philip E Lapinski Thanks a lot.
1.Could you share what was the number or concentration of T cells (were they mouse T cells?) you had used? Was the treatment performed in 12 well or 24 well plates?
2.Also, did you stimulate your cells with anti-CD3/CD28 or maintained them in unstimulated state by adding IL-7 post addition of 4-OH tamoxifen ?
3.I need to use the a large amount of mouse T cells for my western blot. Therefore, could you share what is the maximum number of cells we can use per well treatment.
4. Is there a recommended stock concentration we need to make? Could I just make some single use dilution as low as 1mg/ml in methanol? Any suggestions for the choice for solvent for further dilutions?
Thank you so much.
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