Dear Madina,

Attached are to publications which cover all your inquires and questions.

Hoping this will be helpful,

Rafik

Dear Madina,

Attached are two publications which cover the answers to your inquiries and questions.

Hoping this will be helpful,

Rafik

Dear Rafik,

Thank you for your response, whoever the documents that you attached are only covering cell lines, and I'm working with mice. I'm looking for the more specific information regarding the dietary Doxycycline application in vivo. 

Thank you for your input Joy. This is helpful.

I have read a paper saying that longterm administration of Dox in water leads to dehydration and loss of bodyweight, since my system is Dox-off, I need to keep mice constantly on Doxycycline, so Dox in food is better in my case. But I guess I would still use water for the pilot experiment, and then if it works, I will order food.  Thanks again.

We also use Doxycycline in the water for mice. We use 2mg/ml doxycycline in 5mM saccharin water.  If you look at figure 1F, you will see the timecourse that we see for our specific transgenic mouse. We see maximal knockdown at 14 days post-administration. However, once we remove the doxycyxline from the drinking water, the effect goes away more quickly, in a matter of days. Please see attached paper for methods. Also, make sure to add the saccharin as the mice will not like the taste of the doxycycline...add to control as well.

Article Tonic inhibition of chronic pain by neuropeptide Y

Check with BioServe in NJ. to order grain-based diets with Dox.

Hi Madina

I'd definitely go with Dox food - we use this routinely. The mice don't like Dox water, even with sugar added. They stop drinking and get dehydrated (see attached paper). Plus Dox is very labile in water, so you have to replace it every couple of days even if protected from light. In contrast, Dox food lasts for about 6 months when stored at 4C, and is fine for weeks at room temp - easy! We use Dox at ~600 mg/kg in standard food and most suppliers should be able to formulate this. I've attached an old diet sheet from Harlan Teklad. Good luck!

Ross

We have had no problems maintaining mice for months with Dox in the water plus 5% sucrose, so perhaps there are some strains that are ok, but others not.  The food option sounds interesting; I wonder how expensive it is?

Thank you all for your responses. 

Jonathan, food with standard concentrations of Dox (such as 200 or 2000ppm) is not expensive and can be ordered in different amounts, however 40ppm has to be ordered custom, and the minimum order is for 10kg, which tends to be pricey (over $300). 

Take a look at the paper that Ross attached. It is a comparison between different ways of Dox application. I guess everything depends on the purpose of a study and what is being induced by Dox and for how long animals are being kept on Dox. If dehydration or sweet stimulus don't interfere with the goal of the study, Dox in water should be fine. 

hi Madina, have you completed your tet-off experiments please? did you dox your mice in drinking water or in the chow please? which one the mice like better? look forward to your response!

Much more practical to use the grain-based diet with antibiotic. Sugar water needs to be changed often (or filtered) to avoid fungal growth. Bioserve is a reliable supplier of tet/dox diets. https://www.bio-serv.com/

it would be greatly appreciated if anyone could inform the maxial tolerable duration of dox diet treatment in mice at 600mg/kg. cheers!

We have been using Dox food at 2000mg/kg for our mice carrying Dox-off  genes to turn down the expression. The mice are fine but 2000mg/kg could not completely stop the the expression. I am going to test 3000mg/Kg. One paper showed they used Dox food at 2300mg/kg. 

Madina Makhmutova Penny E Shockett Xiaochong Wu

Can you comment on the biosafety and IACUC requirements for the dox diet? We are trying to implement this in our lab but i'm having a hard time trying to find this info.

Thanks

I use the 625mg Dox food and it is sufficient to block expression in my tet off system. But if you are still seeing expression with dox on I'd recommend that you confirm that your TRE promoter is not leaky. We have noticed that the TREtight promoter will Express even in the absence of tTA but only in certain cell types.

Dear Raj,

I also have observed leaky expression of my transgene of interest under the TRE promoter, in the presence or absence of rtTA (no doxycycline) in vivo in mice. The transgene is about 10-fold higher in TRE+/- mice (with or without rtTA) when compared with the transgene expression of TRE-/- mice with or without rtTA (no doxycycline). I am wondering what the strategy would be to avoid the leaky expression if that is even possible.

If that is not possible, the TRE-transgene design is a failure because the transgene is expressed regardless (not regulated by induction).

Thanks.

Hello xiaobo,

Short answer, yes. There is a way to alter the TRE to make a leaky tre nonleaky.

In our hands we notice a cell type specific leak in the TRE. Fortunately for us, the cell type of our interest did not have the leak.

That being said, my PI has begun modifying the TRE to alter the leak and we have had success invitro. We hope to test this in vivo soon.