We are creating a gene conditioning knockout mice strain by Cre/loxP. I think the first step is to examine the physlogical difference between it with WT. What parameters should I examine? such as weight, food intake, vital organ histology? anything else?
Thanks so much.
I believe the first step after you've confirmed the genotypes would be to confirm that your cre is working, so molecular characterization of your line. Once you are sure about this, then depending on what you have knocked out, you could characterize the phenotypic traits associated with your gene of interest.
You have different steps of control to perform before the characterization of your mouse strain. As said by Agnes above, you have first to control that your Cre is expressed according to the regulatory element you used to drive its expression. It can be done by crossing you Cre line with a so-called reporter strain, e.g. Rosa26-YFP. You also have to be sure that your floxed allele is correctly excised by the Cre recombinase, so a bit of tissue specific PCR genotyping is in order (dissect out some tissues and check that your floxed allele is excised where it should be and not excised where it shouldn't be). Then you have to be sure that either Cre alone or your floxed allele alone don't have any influence on your phenotype of interest (although it can be done in the course of your physiological / phenotypical characterization of the line). Anyway, I guess that you made this strain for a particular purpose, so what parameters you should look at depends on your biological question. Good luck!
After the indicated controls I would suggest that the basic charcaterization includes: a) survival kinetics; b) weigth; c) histopathological analysis of all tissues e.g. in 2-3 month-old mice; d) check fertility by stablishing crosses; e) basic behavior observation (neuromuscular coordination, learning and memory)... Obvioysly, as also indicated above, this very much depends on your biological question and the known/expected activities of the gene..
I think that you have created some kind of gene conditioning KO mice, and have checked the genotype of mice that you are interested.
Now you want to look for the phenotype on those mice.
1.Preparing for BrdU labeling, it needs only to make one injection of BrdU at one hour before killing the mouse. It can be used to check the proliferating function of every organ you are going to take and at any time you want to check by immunohitochemical (IHC) stain on paraffin or frozen sections.
2.At first you can take more tissues for screening the possible lesions in different organs.
Later on, you can decrease the number of organs without specific findings.
Every organ you take must be cut into two parts. One part fixed in formalin for routine HE and IHC staining. Another part of tissue should be frozen in OCT mounting medium for some special staining or some molecular analysis.
Mei Ling
If the conventional knockout mice are lethal or if not, I'd like to use CMV-Cre to delete the gene to test the function (if lethal) and confirm gene deletion.
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