We are trying to generate a CKO mouse model for a protein of interest. And we plan to use Crsipr/Cas9 to flox the full length of the gene, which is more than 20k bp. It seems most of the CKO mouse model out there are targeting one or a few exons. So I wonder, does the size of floxed DNA fragment reduce the efficiency of Cre-mediated recombination? If yes, what is the maximum size that Cre is able to work on?
There has been some reports suggesting Cre-lox system can induce inter-chromosomal recombination (Serth et al., 2015; Koike et al., 2002., etc). It seems that the size would't be a problem. But those studies are done on mouse embryo or cells, what about young or adult mice?
Any insight is greatly appreciated. Thanks so very much in advance.
Zhen
Yes it does affect the efficiency of recombination even at the level of a few tens of base pairs difference, unless you have a good reason to delete the whole gene, just delete one of the early exon(s) to produce a null allele. I don't have specific references for you.
I'm not sure if this helps, but page 3 of the protocol linked below states: "The floxed region should ideally be 2 kb or shorter--that is, up to 2 kb between the loxP sites."
https://ncifrederick.cancer.gov/research/brb/protocol/protocol4_cko_design.pdf
In the following article, I found that the rate of Cre recombination in S. cerevisae depends critically on the length of floxed region. Supplementary material, Figure S7.1.
Preprint A light tunable differentiation system for the creation and ...
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