Hello, I am doing a study on lineage tracing using a cell-type specific reporter crossed with a CreER line. I am looking to study these at e17.5 and am curious if anyone has recommendations for when/how much tamoxifen I should inject to the adult mother to activate it? I have been looking at other papers and am mostly finding results for postnatal injections for which it is once a day for five days leading up to the study. I do not have much experience working with mice so do not really have experience using the CreER system other than reading about it. Thanks!
Hi Nathan,
actually there is quite some variability with Tamoxifen IP injections. You can inject the pregnant mother with between 0.5 and 10 mg/30 g. Tamoxifen has a half life of about 48 hrs, and normally you can see the deletion in two days from injection. When you want to delete, depends on when your gene is expressed and therefore how long you want the embryos to develop without your gene.
But, there are two issues.
Solubility of tamoxifen is not trivial. You can solubilize it in corn, peanut or sunflower oil (pre-warmed at 65 C is probably better), some people add 10% ethanol that is not toxic but helps. You'll have to keep it warm at 65 C (if you use ethanol, 42 C should be enough) for hours to have a complete solubilization and protect from light. Some people sonicate, if you have a sonication bath that would do, switching on the US once in a while for a min or two... Solubility is up to 20 mg/ml, but dissolves more easily at 10 mg/ml. Anyway, keep in mind that injecting this dense oily solution is not easy, you have to be sure you are truly intraperitoneal and go slowly. Try not injecting more than 200 ul (I know, this reduces the max amount of Tamoxifen you'll actually be able to inject...). Consider also administration in drinking water, I copy from a friend:
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.
(TAM injections in the CreER-LoxP system?. Available from: https://www.researchgate.net/post/TAM_injections_in_the_CreER-LoxP_system/1 [accessed Aug 15, 2017].)
Second issue, more important, is your specific deletion. Every locus has it's own deletion kinetics, and what works for one flox allele/line often does not for another. So you should try 2-3 different amounts, say 0.5, 2 and 4 mg (so you stay within 200 ul injection volume at 20 mg/ml) at E15.5 and check your deletion at E17.5. If there is no or too little deletion you'll have to adjust. You can inject earlier and do multiple injections every two days, or every day for 3-5 days. If wt embryos are affected in development then you have to reduce the dose, it means it's being toxic... so you can do multiple injections at low dose.
Good luck!
When injecting pregnant females, weigh them and I inject 5mg/40 grams maternal body weight of tamoxifen dissolved in corn oil (20mg/mL). This gives a high rate of recombination so it may need to be tailored if you are trying to achieve a very narrow labeling window. (Ref: Srinivasan et al., Genes & Dev, 2007).
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