I have a Cre driving loxP recombination in some neuronal precursor cells. I crossed the Cre to 2 conditional KO alleles for 2 different genes to generate a double conditional KO mouse (X-Cre; GeneA lox/lox; GeneB lox/lox). However, in the end, in the cells that contain all 3 elements, GeneA was nicely knocked out, while GeneB only achieved knockdown of 75%. Usually, when people refer to an "incomplete knockout", it has something to do with Cre expression. But in my case, it seems like Cre expression is fine since GeneA is knocked out. Is there anyone who can provide a possible explanation to this please?
I guess you have a problem with accessibility of the locus. It is possible that your GeneB in a specific developmental condition of the cells is not accessible by the Cre because chromatin is closed (heterochromatin) around it. The LoxP recombination system typically works in conditions of active transcription of the targeted gene. Since your brain extract averages over precursor cells at different developmental stages, you find the deleted and the non-deleted conditions at the same time. Most likely in vivo you will have to live with this because you will never be able to synchronize your precursor cells. But you might try in vitro.
I guess you have a problem with accessibility of the locus. It is possible that your GeneB in a specific developmental condition of the cells is not accessible by the Cre because chromatin is closed (heterochromatin) around it. The LoxP recombination system typically works in conditions of active transcription of the targeted gene. Since your brain extract averages over precursor cells at different developmental stages, you find the deleted and the non-deleted conditions at the same time. Most likely in vivo you will have to live with this because you will never be able to synchronize your precursor cells. But you might try in vitro.
Assuming that you used identical flox sequences, it is going to be due to context. Some floxed alleles are highly sensitive, others not so much so, so you can get differential excision even with the same Cre.
How about the Knockout efficiency of GeneB lox/lox;Cre mice? If it is also 75%, explanation will be easy.
I recommend the following: take this 2nd gene, have flox/wt mouse ,cross to Cre driver, which is expressed in zygote/early blastomeres, to pass "Null" allele through germline. Then generate flox/null, Cre mice. It is expected that the excision will be higher because only 1, not 2 alleles have to be converted to null. We stopped crossing to ROSA26 -stop.. reporters as ROSA26 always works and may not be a good indicator/predictor for excision of your floxed gene (this is what you want), only as a guideline on how your Cre works in the ideal world. Agree with previous comments.
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