Hello everyone,
I've been struggling for a while with two Tamoxifen dependent Cre expressing mice lines.
I breed both strains with the reporter line Rosa26. Have done extensive genotyping to verify the presence of both Cre and tdTomato in the offspring.
I've injected (i.p.) 100 microL per day, for 5 consecutive days, of Tamoxifen in corn oil at 20mg/L. To prepare it I dissolved the Tamoxifen either directly in corn oil, or in 10% (of final volume) ethanol, then added 90% corn oil, then used a speed-vac to evaporate the ethanol before injection. The Tamoxifen was then kept at -20°C until administration (no more than 5 days).
And yet, after waiting 7-10 days after the injection for expression, when I look at the brain slices I get very inconsistent results:
Some animals have cells market in tdTomato as would be expected, others have absolutely nothing. Some animals have a slight leak (even without Tamoxifen administration), others have absolutely none.
I thought that my injection technique might be the issue, as I understand that if accidentally injected above the peritoneum and below the skin, instead of into the peritoneum, the oil will not dissolve. So I followed up and after performing the perfusion I've checked the injection sites. I have seen some deposits of oil, but not in an amount or size that would account for the amount of oil I've used, leading me to conclude that most of it has indeed been properly injected and absorbed, delivering the Tamoxifen as proper. In any case, the presence and size of such oil residue didn't seem correlated with expression of tdTomato, which seemed to be all-or-none.
This is a bottleneck in my work and I'm having a lot of difficulty with coming up with things to try, any advice would be greatly appreciated.
Thanks,
Ronni
If you are using Tamoxifen free base, it has very poor solubility and should be made fresh every time. I make it to 100mg/ml by first heating at 70C in 70% ethanol, then diluting with sunflower oil (also preheated to 70C). I dose this by oral gavage.
Regarding successful cre recombination: Keep in mind, Nestin is predominately a developmentally expressed protein, there is very little expression of Nestin in the adult brain (subgranular zone), so you wouldn't expect to see many positive cells.
Regarding "leaky" cre (cre recombined cells in mice that weren't dosed with tamoxifen) - If you have untreated mice in the same cage as treated mice, there will be some exposure of littermates to tamoxifen even if they weren't dosed. If you have vehicle or untreated controls, they should be separated from tamoxifen treated littermates. I usually separate them until 2-3 days after the last tamoxifen dose.
Thank you for your response Aaron,
I do prepare the Tamoxifen fresh every time, I find that heating is not needed, and I just suspend it in 100% ethanol and then mix that with corn oil and vortex for 10-15 minutes.
I am aware that Nestin is not abundant, but am using this line specifically for the targeting of the sub-granular zone, so that was where I was looking at at all times.
As for the leak, interestingly I did not house treated animals with untreated ones. Yet there were occasions in which completely naive animals, housed only with other completely naive animals, expressed some leak recombination. To further complicate the issue this occurred simultaneously with two lines (Nestin and Gli1) in one generation, but not again in either the following. Still have no explanation for that.
Finally, regarding the original issue I raised - It appears that the problem was the administration route. Despite it seeming as if the i.p. injections were absorbed just fine in most cases, once I attempted dosing by oral gavage there was good recombination across the board.