Cre lox Tet-On Transgenic Mice... - where to start? (Jul/13/2005 )

Good Day,

I am confused on how to start. I understand how the cre/lox system works, and how you can further fine tune it with the tet system.

I want to knock out a certain gene (in certain tissue), when I feed the mice doxycylcine.

Exactly, where do I start? I know what gene I want to knock out.
I assume I clone that gene into an expression vector (like pcDNA3.1)
How do I get that expression vector into a mouse? Inject it into a fertilized egg?

Any good references on this would be GREATLY appreciated!!
Thanks very much,
a non-life science grad student

katie, there are people doing this in my lab.. I am not one of them.. but I can tell you that this is a difficult project. Popping your gene of interest into pcDNA3 and injecting it into a mouse egg is not going to do it.

I don't want to be discouraging, but this is something a person should do under the trained supervision of a person who has done it successfuly more than once. Making a knockout mouse it no trivial project.

I am afraid it is going to take more than reading reference to make this happen.

I am totally serious.

Hi pBluescript,

Thanks very much for your advice. I should have clarified: I am learning how to do this soley for the purposes of a graduate school exercise. I have to create a protocol for an experimental approach I propose. I am not physically going to caryout the actual experiment.

I appreciate your concern, because I am evaluated on my experimental approach.

Do you know of any good references that discusses troubleshooting or limitations when creating cre/lox transgenic mice? I am unfamiliar with life-science related experiments.

Thanks very much for your wisdom,
Katie

Try reading this paper then click on related articles- should give you everything you need.

http://www.ncbi.nlm.nih.gov/entrez/query.f...5371&query_hl=2