CD11c.DTR Mice die after single D.T. treatment - (Aug/24/2011 )

Thanks in advance for anyone who answers.

I've recently been using CD11c.DTR mice, which express simian diphtheria toxin receptor under an Itgax promoter (drives CD11c expression). This makes cells that express CD11c sensitive to diphtheria toxin treatment in transgenic mice, most of which are dendritic cells, but also a few NK cells and a few CD8 memory T cells at low levels. However, I've been running into a brick wall with my research in that my mice die usually within a week to 10 days after diphtheria toxin injection, and I need them to lives for months. I can't find any information on any forum or protocol that describes a similar situation. Many papers even exist where this line of mice was injected with D.T. and allowed to live for months without problems. Here are some details of what I've been doing.

-The mice are bred as CD11c.DTR+ heterozygotes x WT C57/Bl6, litters are generally 50/50 WT:het (so no developmental problems)
-I've been giving a single dose of Diphtheria toxin, which is fairly standard...5ng/gram of body weight given I.P.
-I have also tried 2ng/gram of body weight on days 0 and 2 with the same difficulties
-CD11c.DTR- mice (WT, basically) are unaffected by DT, and do not have any apparent phenotype in response to D.T. treatment

A few side notes on the issue...
-A post doc in a neighboring lab tried injecting 4ng/gram of body weight in the footpad. He used our D.T. stocks and some of our mice. His mice also died. At very low doses (not specified to me, but I'm guessing 1-2ng/gram of body weight) in the footpad, he said they sometimes lived, but looked really bad for a week or so.
-By chance, I did find another lab with a similar problem. They switched their mice to a liquid diet (gel-like food), and injected them with saline to help them live, but apparently many of their mice still died a couple weeks post treatment
-I am aware that it is typical for CD11c.DTR+ mice to not survive more than 2 doses of D.T. over a week span, so I know these mice CAN be overly sensitive to D.T. treatment

Thanks for reading my novel, and I'd be grateful of any information anyone can give on the matter.

Are you sure your dosage is correct (e.g. not supposed to be ng/kg? Did you dilute/dissolve it yourself? Is this dose appropriate for these mice? Does the expression of the receptor make them more susceptible?

bob1 on Thu Aug 25 01:28:44 2011 said:

To answer these questions, the dosage is correct (4-5ng/g). Normal mice are not susceptible to the toxin. Only cells that express the simian toxin receptor are susceptible to the toxin (mostly dendritic cells). The one thing I cannot verify is that the toxin is at the concentration that it was labeled as when it was made by a previous lab member, who I believe is competent.

It sounds like the CD11 specificity is not so specific after all, or that the mice are particularly susceptible to DT. What dosages have other labs that have used these mice been using? Perhaps you need to try titrating the dosage - e.g. start at 4 ng/g and work down to 0.1 ng/g and see if you still get the same response, and that your cells are triggered.

bob1 on Fri Aug 26 05:14:56 2011 said:

I think that will be my next course of action. These mice are very commonly used, and the dosage is standard. I will also try to keep mice alive giving saline injections every day or two post D.T. injection.

Anyways, I appreciate the input, and I certainly will continue checking this post to see if anyone has any other ideas.

Hi,
As you pointed-out this is actually a well known side effect of DT treatment on CD11c.DTR mice published initially by Jung & Lang (immunity 2002, 17:211-220). The only way to go around that (has been published many times) is to work with BM chimeras (CD11c.DTR BM into wt host). One thing you probably have to consider also, is that DT efficiency and off-target toxicity can greatly vary from batch to batch. In my experience DT from Sigma has to be banished ...