Please, check this publications. Sometime, the reporter (GFP, Cherry...) can be important.

Non-parallel recombination limits cre-loxP-based reporters as precise indicators of conditional genetic manipulation

How many generations did u have from this matings??

@ Antonio: Thank you for the reference, we work with a lot of reporter mice and lineage tracing and that is a very interesting and potentially terrifying read.

@Kylie: In addition to Antonio's reference, your idea that regulatory elements are different in these different tissues occurs elsewhere as well. Olfactory epithelium which contains multiple neuronal progenitors and stem cells, a large portion of drivers and transgenic mice do not work in the adult due to special gene regulatory systems which are in place to assure that each olfactory sensory neuron only express a single olfactory receptor.

A couple observations, however: your driver mouse is a knock-in IRES construct, and not a bac transgenic. Therefore, any regulatory effects on the driver itself are the same or nearly the same as the wildtype. However, this also means that you will get potentially better efficiencies if you can breed them to homozygote status. The PCR strategy for genotyping het/homo does exist, so it may be worthwhile, if you haven't done so already.

Second, because IRES is dependent on a separate ribosome binding event, you are not even remotely guaranteed of a 1:1 ratio of Cre to Chat. Different cells have different efficiencies of IRES use, ranging from 6% all the way to nearly 100%. You may be unfortunately studying a cell type which does not like IRES very much. This doesn't apply for this particular driver, but IRES activity seems to also vary with age, younger mice exhibiting more IRES efficiency, so in conditional mice, you could induce with tamoxifen at a younger age.

Lit Ref: Absence of Internal Ribosome Entry Site-Mediated Tissue Specificity in the Translation of a Bicistronic Transgene

http://jvi.asm.org/content/73/4/2729.full

I know well how frustrating this is, as my entire field is basically crippled at studying adult neurogenesis because a ton of drivers do not work in our tissue and we have to painstakingly make directed knock-ins using more difficult but much more efficient reporter cassettes. Really...it just sucks. Good luck in your future studies though!

Hi Cane,

First look recombination at DNA level. You can use PCR method to check your Cre activity from different cells using DNA. Please contact, if any.

Shab

I have similar problem.

I am crossing the B6.129-Nos1tm1(cre)Mgmj/J mouse and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse(Ai14), both from Jackson lab. The expression of tdTomato in cochlea nuclei don't really overlap with immunestaining of Nos1. Some cell types are only labeled with tdTomato and some types are only with antibody,