We have experienced significant difficulty when inducing the recombination of a CreERT2 Tg mouse line in fetal liver for lineage tracing purpose. CreERT2 mice were mated with R26R-EYFP report line.
Various injection doses were attempted, for instance, 1mg/injeciton/day Tam intraperitoneal injection to the plugged mother at E8.5-E11.5 and E12.5 embryos were harvested for flow analysis of the presence of YFP+ve cells. The absolute number of YFP+ve cells in the fetal liver were extremely low and it is about ~0.1% of total liver cell suspension (We are expecting to label 1-2% of total liver cell). We used cells harvested from embryonic limbs of the same embryo for positive control and limb cell suspension contains >15% YFP+ cells. Thus we knew that our Tam injection went into these embryos. In order to examine whether we have injected sufficient amount of Tamoxifen, we have also tried to inject 2mg Tam/injection/day and 1mg Tam/injection twice a day. Neither of them make significant difference in the number/percentage of YFP+ cells in fetal liver, however, higher injection doses do increase the percentage of YFP+ in embryonic limb upto 30%.
After I searched in Pubmed, I couldn't find a lot of successful Cre recombinations in mouse fetal liver other than Hayashi's work (PMID: 11944939). In his work, he shows there is a marginal labelling of fetal liver cells when injecting 9mg tamoxifen.
Does anyone have experience with inducing CreERT in fetal liver? Or this is known to be challenging?
Eric,
The Tamoxifen concentration may still be too low. Our lab typically makes a 20mg/ml solution of Tamoxifen and injects 5mg of Tamoxifen per 40g body weight of the mouse. We don't study fetal livers (yet), but I did manage to find an article from a group that had success with CreERT2 in this system (PMCID: PMC3494970).
They used a similar approach. Briefly, they performed a single intraperitoneal injection using a concentration of 4mg/40g body weight at days E14.5 - E16.5 and harvested the embryos 18 hours later. The later timepoints could make a difference but I'm unsure if that's the case.
Hope this helps.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494970/
Dear Eric,
Using Tamoxifen can be easy but can be a painful experience as well.
To start up with make sure you will not cause embryonic death. You really have to time the embryonic development before the Tamoxifen administration. For instance, I had to inject my mice at 2 a.m to be sure the embryos would be at E9.5. Before that I would cause embryonic death due to hearth malformation. We even had a light-cycle reverted box to make my life easier at the end.
OK, the dosage. I tried several dosages but a final dose of 1 mg should work. Make sure you pay attention to the animals body weight. Unless you're working with giants 1 mg should be fine.
Regarding the Tamoxifen administration periodicity it is really locus dependent. we published 2 papers with a single dose of Tamoxifen to knock-down Bmp2, Bmp4 and Bmp7 in pregnant females (embryos at E9.5) and my latest project required 5 consecutive injections on 10 days old pups.
The result with the Bmp projects were easy to assess, the mice would have no kidneys or digits were absent. The project where I had to inject the pups (also tried to inject the mothers and the result was the same) resulted in almost 100% depletion of the gene product. The problem was that we could not observe tumor formation. The tamoxifen worked well but the gene was just not responsible for tumor formation.
As a rule of thumb, with 1 mg you shouldn't go wrong.
Good luck, Eric
Thanks Tim and Alex for your input. We need to harvest those embryos at no late than E13.5 so I thought 1-2mg Tamoxifen should be plenty because those embryos are still quite small. As Alex pointed out, excess amount of tamoxifen will lead to increase the chance of embryos been reabsorbed. I reckon I will first try another CreERT2 locus, Nestin, which should be expressed by more cells in the liver than my target locus.
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