Our protocol for dissolving tamoxifen is similar to Jan's above, but we use Corn Oil from Sigma rather than Sunflower oil. We make our stock at 10mg/ml, which is frozen at -20C until use. The tamoxifen is thawed at 37C for 15 minutes before use. We give the mice 2 IP injections on subsequent days, at a dose of 0.2mg/ gram body weight. This dose is sufficient to delete our gene of interest nearly completely in all tissues of the adult mouse.

I have tried to do tamoxifen administration to pregnant female mice (at approximately E18), but have always encountered unacceptable levels of fetal death. The pups are usually born, but die after 1 or 2 days. I have heard that fostering the newborn pups to a different mother can help mitigate neonatal death, but haven't tried it myself.

As for the deletion efficiency, this depends very much on the individual gene that you are attempting to delete, some floxed alleles work better than others with the same Cre transgene. It is possible that you just got unlucky, and your gene(s) are not deleted well by the Cre.

For our protocol, please see the following references:

J Clin Invest. 2012;122(2):733–747

Genesis. 2007 Dec;45(12):762-7

As Jan suggested, add EtOH to dissolve TAM powder, and then mix with sunflower oil. I think this would be much better than sonication in oil.

This website might be informative.

http://openwetware.org/wiki/Tamoxifen_administration_to_mice

Best regards,

Naoto

http://openwetware.org/wiki/Tamoxifen_administration_to_mice

Hi Daniela,

Did the genetic background of your mice change? Also, it might be worth a try to gavage the Tamoxifen instead of injecting it. You can deliver higher doses of Tamoxifen and we found it to be better tolerated than ip injections.

Best,

Stefanie

Hi there.

one more suggestion would be to aliquot your tamoxifen solution not to freeze-thaw it multiple times. Also protecting it from light is a good practice in the long term.

For better resuspension, this is our protocol:

1. Tamoxifen is suspended in peanut (or corn) oil to a final concentration of 10 mg/ml. Note that Tamoxifen does not readily go into suspension, so it is recommended to either use a pestel to accelerate its resuspension or place the tube on a rotating wheel overnight. The tube should be protected from light.

2. Aliquot the solution in 1.5 ml tubes and store them at -20 ˚C.

3. Before injection, thaw the required aliquots and bring them to room temperature or, preferably, warm them up to a temperature close to that of the mouse.

Alternative to the IP injection, you might consider administration of tamoxifen in chow or drinking water.

Administration in chow:

Keep in mind that dosage in this case depends on eating behavior. Tamoxifen formulation is commercially available. The standard concentration provided is 400 mg of tamoxifen citrate per Kg of irradiated food pellets (Kiermayer et al. 2007).

Note: Mice might suffer a temporary loss of weight immediately following transfer to the new diet.

Administration in drinking water:

Tamoxifen can be first dissolved in pure Ethanol (i.e. 100 mg tamoxifen in 1 ml of ethanol) and then diluted 100-200 fold in distilled water (final concentration 1-0.5 mg/ml). Mice drink an average of 4-5 ml of water per day.

Hope this helps!

Laura

If it was working before, than either your mice changed or you're tamoxifen is no good. Since your lab mate is having the same problem, I suspect your solutions. Buy a new bottle, weigh it out fresh, and as described above, store in aliquots in the dark. Did the solution heat up when you sonicated it? You may have inactivated it. Try putting it on a rocker in the cold room instead. Finally, find someone with a lacZ or other reporter line that you can quickly test your solution on. if the solutions are good, your Cre must be "dead"

Hello and thanks to everybody for your responses! I will try to answer most of what came up here:

The TAM I used that didn't work came from a new bottle I just opened, as opposed to the one I had been using, that was from a different bottle. Nevertheless, I checked the Lot No. and it's the same as the previous bottle, so I'm thinking I may have a problem with this particular Lot. I give you the number below, in case anyone has this lot and has noticed problems with it:

Cat No. T5648-1G

Lot #: SLBB0439V

PCode: 1001372894

I used to sonicate the TAM in a refrigerated room but now the sonicator has been moved somewhere else. I wish I had written down when I sonicated in the cold room so I could check if those were the times when the drug worked at least a little bit. I will try to avoid sonication like some of you suggested by dissolving it rocking in the cold room away from the light. Since some of you do use heated oil to dissolve it, I'm not sure of the temperature would be a problem here or not. Of course high temperatures would inactivate it but I would have noticed if the sonicator had risen the temperature of the oil a bit too much.

I am also going to try to dissolve it in EtOH before dissolving it in oil to avoid sonication. Does anyone have a suggestion on how much EtOH is needed to dissolve the Tamoxifen? I am concerned wether injecting ethanol will have an effect on embryogenesis or not. There is some literature that goes on this subject, suggesting ethanol does have effect on embryo development so I'm not really sure if I should use it to dissolve my drug.

I always aliquot the TAM solution because working with a big stock is rather uncomfortable so thawing cycles aren't a problem there. I make the stock 10mg/mL too, and the dose I had been injecting (that worked perfectly) is 4mgTAM/30g body weight. (That translates to around 0.13 mg/g body weight). I think I can rise the dose a little bit and see what happens, but I don't want to inject too much liquid via ip, my max is 500uL, so I either make my stocks a bit more concentrated or I rise the dose just a little bit.

The injection I have to give the pregnant mice should be only one, since I need to delete my gene at a given time during embryogenesis and no later than that. The injections are quite early, I've been around 7,5 and 10,5 dpc. I sacrifice the pregnant females at around 12,5 dpc so I don't have issues with postnatal death. I'm only concerned with how easily mice loose their pregnancies when injected. This is another reason why I don't want to bother them so much with double injections or high injection volumes. I have also noticed that they tend to lose more embryos on the side where I injected them, so the TAM does have an effect on wether they lose their embryos or not.

The genetic background of the mice has not changed greately. It was never a pure breed but I've been breeding them myself since I got here so I know they're as pure as they can be. There was no cross with another breed.

I have no experience gavaging mice. Does the drug get to the embryos as fast as it does with an IP injection? Since I need my deletion to be very time constraint, I need an administration route that will deliver the drug to the embryo as fast as possible.

Chow or drinking water are, therefore, not an option. It is for adult mice, though, and I will try this instead of the subsequent IP injections when I work with the adults because I think it's a lot less stressful for the animals. It is a good suggestion, thank you very much!

And lastly, but not least, we did try a reporter system and we did not get the expected recombination so I'm also doubting the solutions myself. We tried more than one solution, in case any of us had commited a mistake. I did it, then my mate did it, and then we did it together. Sometimes it works half ways, sometimes it doesn't work at all. Either it's the drug or the way we are dissolving it, so the suggestions on how to dissolve it will be helpful. I will try those ways to dissolve it and hopefully i won't deactivate it.

Thank you very much everyone for your responses, and I know my answer got too long, but since there's a lot that could be going wrong, I got a lot of good suggestions and I needed to reply!

- Daniela

Good luck!

We dissolve tamoxifen as follows:

1. Warm 1g tamoxifen to room temperature for 30 minutes

2. Warm 10ml 100% EtOH to 55C.

3. Add the EtOH to the tamoxifen and shake well. Most of the tamoxifen will dissolve.

4. Add the EtOh/tamoxifen solution to 90ml of corn oil (sigma). Final concentration is 10mg/ml.

5. Incubate at 37C for 15 minutes, then store tubes at -20C.

6. To thaw tamoxifen, incubate at 37C for 15 minutes.

I don't think gavage is necessary, many groups use IP injection for deleting genes in embryos.

I hope this helps.

As you can see from the answers you have already gotten, there are several ways to deliver TAM and likely from many lots and from many companies. If you have a good Cre, it will likely work with many of these different methods. I personally have used one bottle of TAM for years and I have also switched lots at times (and even manufacturers) and not noticed any striking differences in efficacy, thus, it seems more likely to me that something else has changed. Are your Cre mice knock-ins or transgenics? If they are transgenics, you could be losing copy numbers as you breed successive generations, even if you are inbreeding. This could definitely result in diminished efficacy and eventual loss of recombinase activity even though your mice are still genotyping as positive for the Cre. Similarly, you could have the same problem if your reporter is a transgenic. If possible, you could go back to your founders, or re-derive if you cryo-preserved any gametes. Or, if you think the problem is in the reporter, you could breed with another line, like the ai14-tdTomato line which is a knock-in and works really well (http://jaxmice.jax.org/strain/007908.html). Best of luck.

My mate used the ai14-tdTomato as a reporter line. It is a wonderful reporter! And she also used different Cre lines so I don't think the problem is there...

Tamoxifen Free Base 1000mg from MP biochemical Cat # 156738

To original amber bottle of TAM add 5ml of ethanol (Gold shield 200 proof)

Heat in a water bath at 55 C for 10-20 min vortex occasionally

Labeled 1.5ml tubes keep at 55C

Ali quote 100ul of ethanol mixed TAM It's very quick precipitating as temperature dropping

Sunflower seed oil cat # S5007 from Sigma heat at 55 C

Add to aliquoted TAM/ /ETON of 900ul of Sunflower oil

You have 1ml of 20mg/ml Tamoxifen

To administer 1mg of TAM I am injecting IP of 50ul for 5 days (or 100ul for 2 days)

Warm up at 37C before inject It's working good

Store at -20 C between injections and after making TAM soln,wrap the tube in foil,or keep in the dark

Daniela, i have been using the same TAM as you, Sigma T5648, and have used it during embryogenesis, single IP up to 9mg/30g at E12.5.Not toxic at this dose, but teratogenic at higher concentrations at this stage.I have always prepared it in Corn oil with 5% EtOH, vortex, and incubate at 37C for many hours, with occasional vortexing until well solubilized. The EtOH greatly helps disolving the TAM, and at this concentration it is not toxic during embryogenesis. I have used at least 10 different Sigma TAM lots over the years with no problem. But Sigma is not very reliable, and with other reagents we had significant problems depending on the lot. Do you have any little remaining TAM from a previous lot that you could test to see if it's really the TAM? Or maybe someone from another lab can give you some?You said you inject between E7.5 and E10.5; do you have the same phenotype regardless of the stage you inject? What CreER line are you using? Keep in mind that what the TAM does is to mobilize Cre to the nucleus and the half life of TAM is very short, about 48hrs. To see if you have ANY recombination , I would suggest you design PCR oligos that amplify the recombined flox allele.

Hello Paula!

I don't have any significant amount of TAM left in any flask, but I'll see if I can weigh some and make a dilution of at least 1 mL. That's a good idea!

Since you used it at higher doses I'll also try doing that as well.

I have a PCR where the band dissappears when there's recombination, so it doesn't allow me to see when there's very little recombination, but it makes a difference if there is some. I have to think how to design primers to see if there is any recombination, thanks for the suggestion.

And no, I don't have the same phenotype, it's milder when I inject later.

Is anyone working with this particular lot of Sigma Tamoxifen?

Hi Daniela,

I am writing a bit late so not sure if your problem has been resolved but here are my two cents.

I am using tamoxifen extensively both in vivo and in vitro. I am injecting 100 ul tamoxifen (20mg/ml) at E8.5. I am harvesting at E10.5 and embryos seems to be doing fine and excision efficiency is good. I am not using particular lot from sigma and prepare tamoxifen in pre warmed corn oil. I keep the solution in 65C incubator for 30 min and store it in 4C for no more than a week.

I have used this solution at different stages and in different cre lines and it is giving good result.

As far as I know, injecting more than 200 ul at one time is not advisable. With 150 ul , I was seeing tamoxifen side effect on my E10.5 embryo (they were smaller but still alive).

Thanks.

Hello Ujala,

Thank you for your answer!

We believe now that our issues are with the TAM itself, the drug. We've been having varied issues from no recombination to embryo death, always using the same dose, but varying the flask of tam and the bottle of oil.

As you say, injecting more than 200uL is in fact not advisable. The dose we need to inject is higher than what you use, though, so that's why we're injecting more than 100uL. I've had like i said toxic effects on embryos as well (I harvested 10.5 embryos and they came out to be 9.5, for example) They're smaller and underdeveloped.

Thanks again,

Daniela

Dear Danijela,

Do you have an update on your progress? We also see some variability with excision, so I would be curious to learn what your experience was.

Bob Dettman

Dear all,

Do you have some experience regarding the Tamoxifen admnistration in females? There is some disadvantage in the use of females, taking in account the hormone cycle and the tamoxifen affinity to estrogen receptors, if you are looking for general neural effects of your KO?

Hello Bob, 

I think the main problem was the TAM we were using. I'm going to start trying the injections again this month, so I'll probably know what happens soon... 

I didn't think you could give tamoxifen in chow - I thought you had to use tamoxifen citrate. Tamoxifen citrate in the diet can (supposedly) lead to hepatic steatosis and other liver issues on the timescale of months, but I never heard of acute toxicity. I would be suspicious if you are using tamoxifen instead of tamoxifen citrate.

Hello everyone,

I would like to know how long is tamoxifen sand in solution in corn oil and under what conditions can I store it?

Thank you in advance.

Clara:

We store 10mg/ml tamoxifen in corn oil at -20C for up to one year.  It is quite stable, and even retains activity over several freeze/thaw cycles.  

Clara,

We usually make enough tamoxifen (20mg/mL) for one week of injection, and for this week we store the solution at 4C.

Hi all,

I am interested to delete a gene in T cells and have been successful in doing so using 5mg/mice (oral gavage) for 5 consecutive days. I wanted to maintain the deletion in T cells for about a month for my experiments.

For this kind of long term deletion (1 month) do I need to give some booster doses after the first week to ensure that my deletion is maintained?

Any suggestions?

Namrata - if you are using CreER to remove part of a coding region that is flanked by lox sites, then once it is deleted, it should stay deleted. If after 5 days of tamoxifen you see a significant reduction of gene expression (or other evidence that the gene is deleted), you should be able to stop giving the tamoxifen.

Hello Bradley,

I thought the same untilI came across this paper Article The role of the LAT-PLC-γ1 interaction in T regulatory cell function

In which they mention

"To delete the floxed LAT in vivo, 1.5 mg tamoxifen dissolved in corn oil

was injected i.p. into mice for 2 d. For long-term deletion, the same dose of

tamoxifen was administered once per week thereafter."