I am confused by the Cre-flox system. For the conditional knockout (Cre-flox) system, Cre is expressed under a specific promoter. It is knocked-in using the homologue recombination, so the original gene should be replaced by the Cre? Does the Cre expression affect the original gene expression?
Hi, if you want to drive the expression of the Cre under the ENDOGENOUS promoter you will replace the endogenous gene with the gene coding for the Cre in one allele. It must be noticed that in this way you have to mantain the Cre in heterozygosis otherwise you knock-out the endogenous gene adjacent to the promoter. Moreover you have to notice that the endogenous gene will be expressed only by one allele maybe lowering its total expression.
Otherwise you can use a synthetic promoter (often a shorter version of the endogenous promoter) to specifically drive Cre expression. This construct (promoter+Cre) can be randomly inserted in the genome or inserted in a specific loci (often the Rosa26 locus) by homologous recombination. In this way you will not lose endogenous gene expression like before and achieve specific Cre expression. Obviously it must be noticed that not always a truncated version of the choosen promoter is sufficient to make Cre expression so specific as that will be with the endogenous promoter.
In a k/o mouse, the endogenous gene of interest is constructed with a floxed allele. Cre is then expressed from a tissue specific promoter, which causes recombination and knockout of the engineered floxed allele.
I appreciate your answers very much!
After searched online, some people said that the Cre can be inserted into the region between promoter and ATG code thus the Cre is controlled under the endogenous promoter without affecting the endogenous gene expression.
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